Peptides and combination of peptides for use in immunotherapy and methods for generating scaffolds for the use against pancreatic cancer and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 15/185,990, filed Jun.17, 2016, which claims the benefit of U.S. Provisional Application Ser.No. 62/182,026, filed Jun. 19, 2015, and claims priority from GB1510771.7, filed Jun. 19, 2015, the content of each these applicationsis herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “2912919-048004_SEQUENCE_LISTING.txt,” created onMay 14, 2019, 28,596 bytes in size) is submitted concurrently with theinstant application, and the entire contents of the Sequence Listing areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Pancreatic cancer is one of the most aggressive and deadly cancers inthe world. In 2012, it was the 12^(th) most common cancer in men with178,000 cases and the 11^(th) most common cancer in women with 160,000cases worldwide. In the same year, 330,000 deaths were reported, makingpancreatic cancer the seventh most common cause of death from cancer(World Cancer Report, 2014).

Pancreatic cancer is not one single cancer entity, but several distinctsubtypes have to be distinguished. Exocrine tumors account forapproximately 95% of all pancreatic cancers and include ductal andacinary adenocarcinomas, intraductal papillary mucinous neoplasms(IPMN), solid pseudopapillary neoplasms, mucinous cystic adenomas andserous cystadenomas. The remaining 5% of all pancreatic cancers belongto the subgroup of pancreatic neuroendocrine tumors (World CancerReport, 2014).

Infiltrating ductal adenocarcinoma represents the most aggressive formof pancreatic cancer and due to its high frequency (90% of allpancreatic cancers), epidemiologic data mainly reflect this specificsubtype (World Cancer Report, 2014).

In 2012, 68% of all new cases occurred in developed countries, withhighest incidence rates in central and Eastern Europe, North America,Argentina, Uruguay and Australia. In contrast, most countries in Africaand East Asia display low incidence rates. Globally, incidence ratesappear to be rather stable over time in both genders (World CancerReport, 2014).

Due to a lack of specific symptoms, pancreatic cancer is typicallydiagnosed at an advanced and often already metastatic stage. Theprognosis upon diagnosis is very poor, with a 5 years survival rate of5% and a mortality-to-incidence ratio of 0.98 (World Cancer Report,2014).

Several factors have been reported to increase the risk to developpancreatic cancer, including older age, as most patients are older than65 years at diagnosis, and race, as in the USA the Black population hasa 1.5-fold increased risk compared to the White population. Further riskfactors are cigarette smoking, body fatness, diabetes, non-0 AB0 bloodtype, pancreatitis and a familial history of pancreatic cancer (WorldCancer Report, 2014).

Up to 10% of all pancreatic cancer cases are thought to have a familialbasis. Germline mutations in the following genes are associated with anincreased risk to develop pancreatic cancer: p16/CDKN2A, BRCA2, PALB2,PRSS1, STK11, ATM and DNA mismatch repair genes. Additionally, thesporadic cases of pancreatic cancer are also characterized by mutationsin different oncogenes and tumor suppressor genes. The most commonmutations in ductal adenocarcinoma occur within the oncogenes KRAS (95%)and AIB1 (up to 60%) and the tumor suppressor genes TP53 (75%),p16/CDKN2A (95%) and SMAD4 (55%) (World Cancer Report, 2014).

Therapeutic options for pancreatic cancer patients are very limited. Onemajor problem for effective treatment is the typically advanced tumorstage at diagnosis. Additionally, pancreatic cancer is rather resistantto chemotherapeutics, which might be caused by the dense andhypovascular desmoplastic tumor stroma.

According to the guidelines released by the German Cancer Society, theGerman Cancer Aid and the Association of the Scientific MedicalSocieties in Germany, resection of the tumor is the only availablecurative treatment option. Resection is recommended if the tumor isrestricted to the pancreas or if metastases are limited to adjacentorgans.

Resection is not recommended if the tumor has spread to distant sites.Resection is followed by adjuvant chemotherapy with gemcitabine or5-fluorouracil+/−leucovorin for six months (S3-Leitlinie ExokrinesPankreaskarzinom, 2013).

Patients with inoperable tumors in advanced stage can be treated with acombination of chemotherapy with radiation-chemotherapy (S3-LeitlinieExokrines Pankreaskarzinom, 2013).

The standard regimen for palliative chemotherapy is gemcitabine, eitheras monotherapy or in combination with the EGF receptor tyrosine kinaseinhibitor erlotinib. Alternative options are a combination of5-fluorouracil, leucovorin, irinotecan and oxaliplatin, also known asFOLFIRINOX protocol or the combination of gemcitabine withnab-paclitaxel, which was shown to have superior effects compared togemcitabine monotherapy in the MPACT study (Von Hoff et al., 2013;S3-Leitlinie Exokrines Pankreaskarzinom, 2013).

The high mortality-to-incidence ratio reflects the urgent need toimplement more effective therapeutic strategies in pancreatic cancer.

Targeted therapies, which have already been shown to be efficient inseveral other cancer entities, represent an interesting option.Therefore, several studies have been performed to evaluate the benefitof targeted therapies in advanced pancreatic cancers, unfortunately withvery limited success (Walker and Ko, 2014). Nevertheless, the geneticdiversity of pancreatic cancer might offer the possibility ofpersonalized therapy, as invasive ductal adenocarcinoma with bi-allelicinactivation of BRCA2 or PALB2 was shown to be more sensitive to poly(ADP-ribose) polymerase inhibitors and mitomycin C treatment (WorldCancer Report, 2014).

Targeting the tumor stroma constitutes an alternative approach todevelop new treatments for pancreatic cancer. The typically dense andhypovascular stroma might function as barrier for chemotherapeutics andwas shown to deliver signals that promote tumor proliferation, invasionand cancer stem cell maintenance. Thus, different preclinical andclinical studies were designed to analyze the effect of stromaldepletion and inactivation (Rucki and Zheng, 2014).

Vaccination strategies are investigated as further innovative andpromising alternative for the treatment of pancreatic cancer.Peptide-based vaccines targeting KRAS mutations, reactive telomerase,gastrin, survivin, CEA and MUC1 have already been evaluated in clinicaltrials, partially with promising results. Furthermore, clinical trialsfor dendritic cell-based vaccines, allogeneic GM-CSF-secreting vaccinesand algenpantucel-L in pancreatic cancer patients also revealedbeneficial effects of immunotherapy. Additional clinical trials furtherinvestigating the efficiency of different vaccination protocols arecurrently ongoing (Salman et al., 2013).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and pancreatic cancer in particular.There is also a need to identify factors representing biomarkers forcancer in general and pancreatic cancer in particular, leading to betterdiagnosis of cancer, assessment of prognosis, and prediction oftreatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor-(-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-Helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell−(CTL−) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated peptides of the invention can act as MHC class II activeepitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-Class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated und thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 161 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 161, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 161 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 161,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1AF depict embodiments as described herein.

FIGS. 2A-2C depict embodiments as described herein.

FIGS. 3A-3D depict embodiments as described herein.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Table 4 are furthermore useful in thediagnosis and/or treatment of various other malignancies that involve anover-expression or over-presentation of the respective underlyingpolypeptide.

TABLE 1 Peptides according to the present invention SEQ Official ID geneNO. Sequence Gene ID(s) symbol(s) 1 FVDTRTLL 1278 COL1A2 2 FGYDGDFYRA1278 COL1A2 3 ILIGETIKI 5742,5743 PTGS1, PTGS2 4 ALDPAAQAFLL 84919PPP1R15B 5 ALLTGIISKA 23165 NUP205 6 ALTGIPLPLI 1017 CDK2 7 ALVDIVRSL3995 FADS3 8 ALYTGSALDFV 1293 COL6A3 9 QIIDAINKV 1293 COL6A3 10VLLDKIKNL 1293 COL6A3 11 ALYYNPHLL 10527 IPO7 12 AQYKFVYQV 5784 PTPN1413 FIDSSNPGL 92126 DSEL 14 FIIDNPQDLKV 5362 PLXNA2 15 FILANEHNV 3843IPO5 16 GLIDYDTGI 667 DST 17 GLIDYDTGIRL 667 DST 18 ALFVRLLAL 7045 TGFBI19 ALWHDAENQTVV 23279 NUP160 20 GLIDIENPNRV 11333 PDAP1 21 GLVDGRDLVIV9943 OXSR1 22 ILSTEIFGV 79703 C11orf80 23 KLDSSGGAVQL 23677 SH3BP4 24KLSENAGIQSL 26064 RAI14 25 LINPNIATV 790 CAD 26 SLYTALTEA 4124 MAN2A1 27TLLAHPVTL 27063 ANKRD1 28 VLDEFYSSL 11321 GPN1 29 YILPFSEVL 2132 EXT2 30YIYKDTIQV 346389 MACC1 31 YLDSMYIML 8754 ADAM9 32 YVDDGLISL 5315 PKM 33FLADPDTVNHL 57231 SNX14 34 FLEDDDIAAV 9945 GFPT2 35 FLFPSQYVDV 9871SEC24D 36 FLGDLSHLL 10945 KDELR1 37 FLNPDEVHAI 81610 FAM83D 38FLTEAALGDA 7980 TFPI2 39 FLTPSIFII 79971 WLS 40 GLAPQIHDL 128239 IQGAP341 GLLAGNEKLTM 3880 KRT19 42 ILSDMRSQYEV 3880 KRT19 43 HLGVKVFSV 1291COL6A1 44 ILAQVGFSV 55117 SLC6A15 45 ILYSDDGQKVVTV 131566 DCBLD2 46TMVEHNYYV 131566 DCBLD2 47 LIYKDLVSV 85016 C11orf70 48 LLDENGVLKL 1022CDK7 49 LLDGFPRTV 204 AK2 50 LLFGSDGYYV 10897 YIF1A 51 LLGPAGARA 255738PCSK9 52 LLSDPIPEV 57521 RPTOR 53 LLWDPSTGKQV 54475 NLE1 54 LTQPGPIASA6374 CXCL5 55 NLAPAPLNA 7035 TFPI 56 NLIGVTAEL 80210 ARMC9 57 RLSELGITQA79801 SHCBP1 58 RQYPWGVVQV 151011, 23176, SEPT10, SEPT8, 55752 SEPT11 59SLSESFFMV 54434 SSH1 60 SLWEDYPHV 9697 TRAM2 61 SMYDGLLQA 51393 TRPV2 62SVFPGARLL 10498 CARM1 63 SVTGIIVGV 57722 IGDCC4 64 TLFSEPKFAQV 84886C1orf198 65 TLNEKLTAL 55845 BRK1 66 TVDDPYATFV 1072 CFL1 67 VIWGTDVNV4173 MCM4 68 VLFDVTGQV 9961 MVP 69 VLFSGSLRL 115908 CTHRC1 70 VLGVIWGV100527943, TGIF2-C20orf24, 55969 C20orf24 71 VLLPEGGITAI 9,904 RBM19 72VMASPGGLSAV 54,443 ANLN 73 VMVDGKPVNL 5879,5881 RAC1, RAC3 74 YIDKDLEYV29102 DROSHA 75 FSFVDLRLL 1277 COL1A1 76 LVSESSDVLPK 100129958,KRT8P44, KRT8 3856 77 RLFPGSSFL 90993 CREB3L1 78 SLQDTEEKSRS 2641 GCG 79VVYEGQLISI 2335 FN1 80 LLPGTEYVVSV 2335 FN1 81 VVYDDSTGLIRL 2898,2899GRIK2, GRIK3 82 ALIAEGIAL 1778 DYNC1H1 83 ALSKEIYVI 515 ATP5F1 84FILPIGATV 6509,6510 SLC1A4, SLC1A5 85 FLSDGTIISV 84916 CIRH1A 86GLGDFIFYSV 5663,5664 PSEN1, PSEN2 87 GLLPALVAL 113278 SLC52A3 88IIDDTIFNL 257641,4864 NPC1 89 KLADIQIEQL 5201 PFDN1 90 KLLTPITTL 1293COL6A3 91 LLFNDVQTL 5339 PLEC 92 YLTNEGIAHL 5339 PLEC 93 SIDSEPALV23420,283820, NOMO1, NOMO2, 408050 NOMO3 94 VMMEEFVQL 9875 URB1 95ALADDDFLTV 4173 MCM4 96 ALAPATGGGSLLL 80830 APOL6 97 ALDDMISTL 7203 CCT398 ALDQKVRSV 4130 MAP1A 99 ALESFLKQV 5591 PRKDC 100 ALFGAGPASI 1806 DPYD101 ALVEENGIFEL 11187 PKP3 102 ALYPGTDYTV 64420 SUSD1 103 AVAAVLTQV10280 SIGMAR1 104 FLQPDLDSL 10514 MYBBP1A 105 FLSEVFHQA 5055 SERPINB2106 FVWSGTAEA 23326 USP22 107 FVYGGPQVQL 91039 DPP9 108 IADGGFTEL1107,1108, CHD3, CHD4, 26038 CHD5 109 ILASVILNV 644538 SMIM10 110ILLTGTPAL 84083 ZRANB3 111 LLLAAARLAAA 2923 PDIA3 112 LLSDVRFVL 53339BTBD1 113 LMMSEDRISL 9945 GFPT2 114 SLFPHNPQFI 80135 RPF1 115SLMDPNKFLLL 197131 UBR1 116 SMMDPNHFL 23304 UBR2 117 SVDGVIKEV 10577NPC2 118 TLWYRPPEL 100422910,1025, MIR2861, CDK9, 51755,8621CDK12, CDK13 119 VLGDDPQLMKV 10629 TAF6L 120 VLVNDFFLV 3646 EIF3E 121YLDEDTIYHL 4144 MAT2A

TABLE 2 Additional peptides according to the presentinvention with no prior known cancer association SEQ Official ID GeneGene No. Sequence ID(s) Symbol(s) 122 MQAPRAALVFA 201799 TMEM154 123KISTITPQI    996 CDC27 124 ALFEESGLIRI   1951, CELSR3,  65010 SLC26A6125 ALLGKLDAINV   5876 RABGGTB 126 ALLSLDPAAV   5591 PRKDC 127ALSDLALHFL  10575 CCT4 128 ALYDVRTILL  11065 UBE2C 129 ALYEKDNTYL  80279CDK5RAP3 130 FLFGEEPSKL  23141 ANKLE2 131 FLIEEQKIVV   6164 RPL34 132FLWAGGRASYGV   3192 HNRNPU 133 ILDDVSLTHL   5245 PHB 134 ILLAEGRLVNL   191 AHCY 135 KLDDTYIKA   7266 DNAJC7 136 KLFPGFEIETV    440 ASNS 137KLGPEGELL   6510 SLC1A5 138 NIFPNPEATFV  11198 SUPT16H 139 SIDRNPPQL  6773 STAT2 140 SLLNPPETLNL    890 CCNA2 141 SLTEQVHSL  79598 CEP97 142SLYGYLRGA   9790 BMS1 143 TADPLDYRL   4928 NUP98 144 TAVALLRLL   9761MLEC 145 TTFPRPVTV   4841 NONO 146 VLISGVVHEI  51360 MBTPS2 147YAFPKAVSV   9123 SLC16A3 148 YLHNQGIGV    701 BUB1B 149 ILGTEDLIVEV 79719 AAGAB 150 ALFQPHLINV  10097 ACTR2 151 ALLDIIRSL   9415 FADS2 152ALLEPEFILKA   7011 TEP1 153 ALPKEDPTAV  22820 COPG1 154 KVADLVLML399761, BMS1P5, 642517, AGAP9,   9790 BMS1 155 LLLDPDTAVLKL   2932 GSK3B156 LLLPPPPCPA   2519 FUCA2 157 MLLEIPYMAA 728689, EIF3CL,   8663 EIF3C158 SLIEKYFSV   3838, KPNA2 645680 159 SLLDLHTKV  27340 UTP20 160VLLPDERTISL   1477 CSTF1 161 YLPDIIKDQKA   5496 PPM1G

TABLE 3 Peptides useful for e.g. personalized cancer therapies SEQOfficial ID Gene Gene No. Sequence ID(s) Symbol(s) 162 NADPQAVTM    10916 MAGED2 163 VMAPRTLVL 100507703, HLA-A      3105 164 YLGRLAHEV    23521, RPL13A,    387841, RPL13AP20,    728658 RPL13AP5 165YLLSYIQSI     64151 NCAPG 166 SLFPGQVVI     23649 POLA2 167 MLFGHPLLVSV     8237 USP11 168 SEWGSPHAAVP      5539 PPY 169 FMLPDPQNI    116461TSEN15 170 ILAPAGSLPKI     29914 UBIAD1 171 LLLDVTPLSL 100287551,HSPA8P8,      3306, HSPA2,      3312, HSPA8,      3313 HSPA9 172TMMSRPPVL     57708, MIER1,     79971 WLS 173 SLAGDVALQQL      9918NCAPD2 174 TLDPRSFLL      2149 F2R 175 ALLESSLRQA       595 CCND1 176YLMPGFIHL    168400, DDX53,     55510 DDX43 177 SLYKGLLSV     25788RAD54B 178 KIQEILTQV     10643 IGF2BP3

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, lung cancer, kidneycancer, brain cancer, stomach cancer, colon or rectal cancer, livercancer, prostate cancer, leukemia, breast cancer, Merkel cell carcinoma(MCC), melanoma, ovarian cancer, esophageal cancer, urinary bladdercancer, endometrial cancer, gall bladder cancer, and bile duct cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 161. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 79 (see Table 1), and their uses in theimmunotherapy of pancreatic cancer, lung cancer, kidney cancer, braincancer, stomach cancer, colon or rectal cancer, liver cancer, prostatecancer, leukemia, breast cancer, Merkel cell carcinoma (MCC), melanoma,ovarian cancer, esophageal cancer, urinary bladder cancer, endometrialcancer, gall bladder cancer, bile duct cancer, and preferably pancreaticcancer.

As shown in the following Table 4, many of the peptides according to thepresent invention are also found on other tumor types and can, thus,also be used in the immunotherapy of other indications. Also refer toFIGS. 1A-1AF and Example 1.

TABLE 4 Peptides according to the present invention and their specific uses in other prolifer-ative diseases, especially in other   cancerous diseases. SEQ IDOther relevant organs No. Sequence (cancer)/diseases   1 FVDTRTLLEsophagus   2 FGYDGDFYRA Pancreas, Breast, Esophagus   3 ILIGETIKIUrinary bladder   4 ALDPAAQAFLL NSCLC, Liver, Breast, Ovary,Esophagus, Urinary bladder   5 ALLTGIISKA NSCLC, Colon, Rectum, Liver,Esophagus   7 ALVDIVRSL Leukocytes   8 ALYTGSALDFVNSCLC, Pancreas, Breast,  Esophagus, Gallbladder, Bile  duct   9QIIDAINKV Breast, Esophagus  10 VLLDKIKNL Pancreas, Gallbladder, Bile duct  11 ALYYNPHLL Esophagus  12 AQYKFVYQV Esophagus  13 FIDSSNPGLKidney  14 FIIDNPQDLKV NSCLC, SCLC, Kidney, Liver, Melanoma, Ovary, Esophagus  16 GLIDYDTGI Brain, Breast  17 GLIDYDTGIRLBrain, Melanoma  19 ALWHDAENQTVV NSCLC, SCLC, Liver, Melanoma,Esophagus, Gallbladder, Bile  duct  20 GLIDIENPNRV Urinary bladder  22ILSTEIFGV NSCLC, Pancreas, Leukocytes,  Breast  26 SLYTALTEA Breast  28VLDEFYSSL Colon, Rectum  29 YILPFSEVL NSCLC, Kidney, Brain, Colon, Rectum, Esophagus, Urinary bladder  30 YIYKDTIQV NSCLC, Colon, Rectum 31 YLDSMYIML NSCLC, Stomach, Colon, Rectum,  Liver, Pancreas, Breast,Gallbladder, Bile duct  32 YVDDGLISL Stomach  34 FLEDDDIAAVBrain, Melanoma  35 FLFPSQYVDV NSCLC, SCLC, Liver, Breast, Ovary, Esophagus  37 FLNPDEVHAI NSCLC, Colon, Rectum, Liver, Breast, Melanoma, Ovary,  Esophagus, Urinary bladder  39 FLTPSIFIIBrain, Pancreas  40 GLAPQIHDL Colon, Rectum, Esophagus  41 GLLAGNEKLTMColon, Rectum, Breast,  Urinary bladder, Endometrium  42 ILSDMRSQYEVUrinary bladder  45 ILYSDDGQKWTV Melanoma  46 TMVEHNYYVNSCLC, SCLC, Kidney, Pancreas, Melanoma, Ovary, Esophagus  48 LLDENGVLKLLeukocytes  50 LLFGSDGYYV Liver, Esophagus  51 LLGPAGARALiver, Esophagus  52 LLSDPIPEV SCLC, Melanoma, Ovary,  Esophagus  57RLSELGITQA Esophagus  58 RQYPWGVVQV Esophagus  59 SLSESFFMVSCLC, Breast, Urinary bladder  60 SLWEDYPHV NSCLC, SCLC, Colon, Rectum, Liver, Ovary, Urinary bladder  62 SVFPGARLL SCLC, Leukocytes, Esophagus 63 SVTGIIVGV Brain, Esophagus  64 TLFSEPKFAQVSCLC, Liver, Urinary bladder  67 VIWGTDVNV Brain, Esophagus  68VLFDVTGQV Stomach  69 VLFSGSLRL NSCLC  70 VLGVIWGVNSCLC, Liver, Ovary, Esophagus  71 VLLPEGGITAI Leukocytes  73 VMVDGKPVNLLiver, Gallbladder, Bile duct  75 FSFVDLRLLSCLC, Esophagus, Gallbladder,  Bile duct  77 RLFPGSSFL Breast, Esophagus 79 VVYEGQLISI NSCLC, SCLC, Pancreas, Breast, Esophagus  80 LLPGTEYVVSVSCLC, Liver  81 VVYDDSTGLIRL SCLC, Brain, Leukocytes, MCC,  Ovary  82ALIAEGIAL Urinary bladder  83 ALSKEIYVI Leukocytes  84 FILPIGATVKidney, Stomach, Breast  85 FLSDGTIISV NSCLC, Colon, Rectum, Liver,Melanoma, Ovary, Esophagus, Endometrium  86 GLGDFIFYSV Liver, Pancreas 88 IIDDTIFNL Stomach, Urinary bladder  90 KLLTPITTLNSCLC, SCLC, Colon, Rectum,  Breast  91 LLFNDVQTLEsophagus, Urinary bladder  92 YLTNEGIAHL NSCLC, Colon, Rectum, Melanoma, Ovary, Esophagus  93 SIDSEPALV Brain, Colon, Rectum, Breast, Urinary bladder  94 VMMEEFVQL Brain, Colon, Rectum, Leukocytes, Ovary, Esophagus,  Endometrium, Gallbladder, Bile duct  95ALADDDFLTV NSCLC, SCLC, Stomach,  Leukocytes, Melanoma, Ovary, Esophagus, Urinary bladder  96 ALAPATGGGSLLL Liver, Melanoma  97ALDDMISTL Stomach, Urinary bladder  98 ALDQKVRSV Brain, Prostate  99ALESFLKQV Colon, Rectum, Liver, Breast,  Urinary bladder 100 ALFGAGPASILiver 101 ALVEENGIFEL NSCLC, Liver, MCC, Ovary,  Urinary bladder 102ALYPGTDYTV NSCLC, SCLC, Brain, Liver,  Prostate, Gallbladder, Bile  duct103 AVAAVLTQV Liver 104 FLQPDLDSL Brain, Liver, Pancreas, Leukocytes, Urinary bladder 106 FVWSGTAEA Brain, Esophagus, Urinary bladder 107 FVYGGPQVQL Melanoma 109 ILASVILNV Prostate 110 ILLTGTPALSCLC, Leukocytes, Breast 111 LLLAAARLAAA Liver, Pancreas 113 LMMSEDRISLBrain, Melanoma 114 SLFPHNPQFI SCLC, Brain, Colon, Rectum, Liver, Melanoma, Esophagus, Urinary bladder 115 SLMDPNKFLLLKidney, Brain, Colon, Rectum,  Liver, Prostate, Melanoma,Urinary bladder, Gallbladder, Bile duct 116 SMMDPNHFLBrain, Liver, MCC, Endome- trium, Gallbladder, Bile duct 117 SVDGVIKEVStomach 118 TLWYRPPEL NSCLC, Melanoma, Esophagus 120 VLVNDFFLVStomach, Colon, Rectum, Liver,  Ovary, Esophagus, Urinary bladder, Endometrium 121 YLDEDTIYHL Stomach 122 MQAPRAALVFABrain, Leukocytes, Urinary  bladder, Gallbladder, Bile  duct 123KISTITPQI NSCLC, Liver, Pancreas 124 ALFEESGLIRINSCLC, SCLC, Colon, Rectum,  Liver, MCC, Melanoma, Ovary, Esophagus 125ALLGKLDAINV NSCLC, SCLC, Colon, Rectum,  Liver, Ovary, Gallbladder,Bile duct 128 ALYDVRTILL NSCLC, SCLC, Colon, Rectum 129 ALYEKDNTYLSCLC, Brain, Liver, Ovary,  Esophagus 130 FLFGEEPSKLPancreas, Endometrium 131 FLIEEQKIVV NSCLC, SCLC, Colon, Rectum, Liver, Melanoma, Ovary, Esophagus, Urinary bladder,Gallbladder, Bile duct 132 FLWAGGRASYGV Liver, Ovary, Esophagus 134ILLAEGRLVNL Ovary 135 KLDDTYIKA Liver, Esophagus, Urinary  bladder 136KLFPGFEIETV NSCLC, SCLC, Liver, Ovary,  Esophagus 137 KLGPEGELLColon, Rectum, Liver, Breast, Esophagus, Urinary bladder 138 NIFPNPEATFVNSCLC, SCLC, Brain, Melanoma 142 SLYGYLRGA NSCLC, Colon, Rectum, Liver, Pancreas, Prostate, Breast,  Ovary, Esophagus, Urinary  bladder 143TADPLDYRL SCLC, Endometrium 144 TAVALLRLL SCLC, Leukocytes 145 TTFPRPVTVSCLC, Colon, Rectum,  Leukocytes 146 VLISGVVHEIBrain, Liver, Melanoma, Ovary 147 YAFPKAVSVNSCLC, SCLC, Kidney, Stomach, Leukocytes, Ovary, Esophagus 148 YLHNQGIGVSCLC, Colon, Rectum, Liver,  Esophagus 149 ILGTEDLIVEVNSCLC, SCLC, Liver,  Leukocytes, Melanoma, Ovary,Esophagus, Gallbladder, Bile duct 150 ALFQPHLINV NSCLC, SCLC, Liver, Leukocytes, Breast, Melanoma,  Ovary, Urinary bladder 151 ALLDIIRSLNSCLC, Brain, Colon, Rectum,  Prostate, Urinary bladder 152 ALLEPEFILKAColon, Rectum, Leukocytes,  Urinary bladder 154 KVADLVLMLNSCLC, Colon, Rectum,  Leukocytes, Ovary, Esophagus, Urinary bladder 155LLLDPDTAVLKL Liver, Melanoma 156 LLLPPPPCPA Pancreas, Urinary bladder157 MLLEIPYMAA Colon, Rectum, Melanoma,  Ovary, Urinary bladder 158SLIEKYFSV NSCLC, SCLC, Colon, Rectum,  Liver, Melanoma, Ovary, Esophagus 159 SLLDLHTKV Brain, Colon, Rectum, Liver,  Leukocytes 160VLLPDERTISL NSCLC, SCLC, Liver,  Leukocytes, Ovary, Urinary  bladder 161YLPDIIKDQKA Brain, Liver, Leukocytes,  Melanoma 162 NADPQAVTMSCLC, Kidney, Ovary,  Endometrium 163 VMAPRTLVL SCLC 165 YLLSYIQSISCLC, Colon, Rectum, Liver,  Melanoma, Ovary, Esophagus, Endometrium 166SLFPGQVVI Brain, Urinary bladder,  Endometrium 167 MLFGHPLLVSVNSCLC, SCLC, Brain, Liver,  Pancreas, Prostate, Ovary 169 FMLPDPQNINSCLC, SCLC, Brain, Liver,  Breast, Melanoma, Esophagus, Urinary bladder170 ILAPAGSLPKI Urinary bladder 171 LLLDVTPLSLLeukocytes, Urinary bladder 172 TMMSRPPVL Brain 174 TLDPRSFLLStomach, Liver 175 ALLESSLRQA Kidney, Breast, Urinary  bladder 176YLMPGFIHL Liver, Leukocytes

The table shows for selected peptides on which additional tumor typesthey were found and either over-presented on more than 5% of themeasured tumor samples, or presented on more than 5% of the measuredtumor samples with a ratio of geometric means tumor vs normal tissuesbeing larger than 3. Over-presentation is defined as higher presentationon the tumor sample as compared to the normal sample with highestpresentation.

TABLE 4B Peptides according to the present invention and their specific uses in other prolifera-tive diseases, especially in other cancerous diseases (amendment of Table 4). SEQ ID NO. Sequence Additional Entities  1 FVDTRTLL Melanoma, Urinary Bladder  Cancer   3 ILIGETIKI OC, AML   4ALDPAAQAFLL SCLC, GC, CRC, CLL, Uterine  Cancer, Gallbladder Cancer,Bile Duct Cancer, AML, NHL   5 ALLTGIISKA Melanoma, Urinary Bladder Cancer, Uterine Cancer   6 ALTGIPLPLI NSCLC, SCLC, CLL, Melanoma, Urinary Bladder Cancer, Uterine Cancer, NHL   9 QIIDAINKVMelanoma, NHL, GC, NSCLC  11 ALYYNPHLL Brain Cancer  12 AQYKFVYQVRCC, Melanoma, Urinary Bladder  Cancer, Uterine Cancer  14 FIIDNPQDLKVBrain Cancer, Urinary Bladder  Cancer, Uterine Cancer  15 FILANEHNVUrinary Bladder Cancer,  Uterine Cancer  16 GLIDYDTGI Melanoma  18ALFVRLLAL Melanoma  19 ALWHDAENQT Brain Cancer, Urinary Bladder  VVCancer, Uterine Cancer  20 GLIDIENPNRV Esophageal Cancer  21 GLVDGRDLVIVNSCLC, Melanoma, Gallbladder  Cancer, Bile Duct Cancer, AML,  NHL  22ILSTEIFGV Melanoma, Gallbladder Cancer,  Bile Duct Cancer  23KLDSSGGAVQL SCLC, Melanoma  25 LINPNIATV Melanoma  28 VLDEFYSSL Melanoma 29 YILPFSEVL BRCA, Melanoma, Uterine  Cancer, AML, NHL  30 YIYKDTIQVRCC, Urinary Bladder Cancer,  Gallbladder Cancer, Bile Duct Cancer, AML 31 YLDSMYIML Melanoma, Esophageal Cancer,  Urinary Bladder Cancer  32YVDDGLISL Melanoma, AML  34 FLEDDDIAAV CRC  37 FLNPDEVHAISCLC, Uterine Cancer, NHL  38 FLTEAALGDA RCC, Urinary Bladder Cancer, Uterine Cancer  39 FLTPSIFII Uterine Cancer  41 GLLAGNEKLTMGC, Esophageal Cancer  42 ILSDMRSQYEV BRCA, Uterine Cancer, Gallbladder Cancer, Bile Duct  Cancer  44 ILAQVGFSV Melanoma  46TMVEHNYYV Urinary Bladder Cancer,  Uterine Cancer, GallbladderCancer, Bile Duct Cancer  47 LIYKDLVSV OC  50 LLFGSDGYYVUterine Cancer, Gallbladder  Cancer, Bile Duct Cancer  52 LLSDPIPEVUrinary Bladder Cancer, AML,  NHL  55 NLAPAPLNA Melanoma  56 NLIGVTAELMelanoma, Uterine Cancer  57 RLSELGITQA Melanoma, Urinary Bladder Cancer, Uterine Cancer, AML,  NHL, OC  58 RQYPWGVVQV Melanoma  59SLSESFFMV NHL  60 SLWEDYPHV BRCA, Melanoma, Esophageal Cancer, Uterine Cancer  61 SMYDGLLQA Melanoma  65 TLNEKLTALMelanoma, Urinary Bladder  Cancer, AML  66 TVDDPYATFV Melanoma  67VIWGTDVNV Melanoma, Urinary Bladder  Cancer, AML  68 VLFDVTGQV Melanoma 69 VLFSGSLRL BRCA, Esophageal Cancer,  Gallbladder Cancer, BileDuct Cancer  70 VLGVIWGV Brain Cancer, BRCA, Urinary Bladder Cancer, Uterine Cancer  71 VLLPEGGITAIBrain Cancer, Urinary Bladder  Cancer  74 YIDKDLEYVUrinary Bladder Cancer,   Uterine Cancer  75 FSFVDLRLLRCC, BRCA, Melanoma, NHL  77 RLFPGSSFL GC  79 VVYEGQLISIGallbladder Cancer, Bile Duct  Cancer, NHL  80 LLPGTEYVVSVBRCA, Gallbladder Cancer, Bile  Duct Cancer  82 ALIAEGIALBRCA, Uterine Cancer  83 ALSKEIYVI Brain Cancer  84 FILPIGATVAML, CLL, CRC, HCC, Melanoma,  NHL, OC, Esophageal Cancer,NSCLC, Urinary Bladder Cancer, Uterine Cancer  86 GLGDFIFYSVNSCLC, BRCA, Esophageal  Cancer, Urinary Bladder Cancer  87 GLLPALVALBrain Cancer, Melanoma  88 IIDDTIFNL Melanoma  89 KLADIQIEQLUrinary Bladder Cancer, OC  90 KLLTPITTL Melanoma, Gallbladder Cancer, Bile Duct Cancer  91 LLFNDVQTL CLL, Uterine Cancer, NHL  92 YLTNEGIAHLUrinary Bladder Cancer  93 SIDSEPALV Melanoma, AML  94 VMMEEFVQLNSCLC, SCLC, Melanoma, Urinary  Bladder Cancer  95 ALADDDFLTVRCC, BRCA, Uterine Cancer,  Gallbladder Cancer, Bile Duct  Cancer  96ALAPATGGGSL NSCLC, Gallbladder Cancer,  LL Bile Duct Cancer, NHL  97ALDDMISTL Melanoma  99 ALESFLKQV NSCLC, RCC, Brain Cancer, CLL, Melanoma, OC, Esophageal Cancer, AML, NHL 100 ALFGAGPASIUrinary Bladder Cancer 101 ALVEENGIFEL Uterine Cancer 102 ALYPGTDYTV AML103 AVAAVLTQV Esophageal Cancer, Urinary  Bladder Cancer, UterineCancer, Gallbladder Cancer,  Bile Duct Cancer, AML 104 FLQPDLDSLSCLC, Uterine Cancer 106 FVWSGTAEA Melanoma, Uterine Cancer, AML,  NHL107 FVYGGPQVQL CLL, Urinary Bladder Cancer,  NHL 108 IADGGFTEL AML 109ILASVILNV Urinary Bladder Cancer 110 ILLTGTPAL Uterine Cancer 111LLLAAARLAAA AML, PrC, BRCA, CRC,  Gallbladder Cancer, Bile DuctCancer, Melanoma, NHL, OC,  Brain Cancer, NSCLC, RCC, SCLC, Urinary Bladder Cancer, Uterine Cancer 113 LMMSEDRISLNSCLC, Urinary Bladder Cancer 114 SLFPHNPQFI NSCLC, CLL, AML, NHL 116SMMDPNHFL NSCLC, Melanoma 117 SVDGVIKEV Melanoma, AML 118 TLWYRPPELCLL, Urinary Bladder Cancer,  Uterine Cancer 120 VLVNDFFLVBRCA, Melanoma, Gallbladder  Cancer, Bile Duct Cancer, AML 121YLDEDTIYHL Melanoma 123 KISTITPQI Brain Cancer, Melanoma,  Urinary Bladder Cancer,  Uterine Cancer, AML, NHL 124 ALFEESGLIRIBRCA, NHL 125 ALLGKLDAINV NHL 126 ALLSLDPAAVBrain Cancer, Urinary Bladder  Cancer, AML 127 ALSDLALHFLCLL, BRCA, Melanoma, Urinary  Bladder Cancer, AML, NHL 128 ALYDVRTILLBRCA, Urinary Bladder Cancer,  AML 129 ALYEKDNTYLNSCLC, BRCA, Urinary Bladder  Cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct  Cancer, NHL 130 FLFGEEPSKLRCC, CLL, Melanoma, Esophageal  Cancer, Urinary Bladder   Cancer, AML131 FLIEEQKIVV AML, NHL 132 FLWAGGRASY Brain Cancer, Melanoma,  GVUterine Cancer, AML 133 ILDDVSLTHL Melanoma 134 ILLAEGRLVNLNSCLC, Melanoma 135 KLDDTYIKA Melanoma, Uterine Cancer 137 KLGPEGELLMelanoma, AML 138 NIFPNPEATFV BRCA, Urinary Bladder Cancer, AML, NHL, OC 139 SIDRNPPQL Melanoma, AML 140 SLLNPPETLNL AML 142SLYGYLRGA CLL, Melanoma, Gallbladder  Cancer, Bile Duct Cancer, AML 143TADPLDYRL Melanoma, AML 144 TAVALLRLL BRCA, Gallbladder Cancer, Bile Duct Cancer 145 TTFPRPVTV HCC, Gallbladder Cancer, Bile  Duct Cancer 146VLISGVVHEI CRC, Uterine Cancer 147 YAFPKAVSVGallbladder Cancer, Bile Duct  Cancer 148 YLHNQGIGVUrinary Bladder Cancer,  Uterine Cancer, AML, NHL, OC 149 ILGTEDLIVEVPrC, BRCA, CRC, MCC, GC,  Urinary Bladder Cancer, Uterine Cancer 151ALLDIIRSL BRCA, Uterine Cancer, AML 152 ALLEPEFILKANSCLC, Brain Cancer,  Gallbladder Cancer, Bile  Duct Cancer 154KVADLVLML Gallbladder Cancer, Bile Duct  Cancer 155 LLLDPDTAVLKLSCLC, CLL, BRCA 156 LLLPPPPCPA Melanoma, Uterine Cancer, Gallbladder Cancer, Bile Duct  Cancer 157 MLLEIPYMAA Uterine Cancer 158SLIEKYFSV CLL, BRCA, Urinary Bladder  Cancer, Uterine Cancer, AML,  NHL159 SLLDLHTKV NSCLC, Melanoma, Urinary  Bladder Cancer, Uterine Cancer160 VLLPDERTISL BRCA, CRC, Gallbladder Cancer, Bile Duct Cancer, Melanoma, Brain Cancer, GC, RCC, Uterine  Cancer 161YLPDIIKDQKA Uterine Cancer NSCLC = non-small cell lung cancer, SCLC =small cell lung cancer, RCC = kidney cancer, CRC = colon or rectumcancer, GC = stomach cancer, HCC = liver cancer, PrC = prostate cancer,BRCA = breast cancer, MCC = Merkel cell carcinoma, OC = ovarian cancer,NHL = non-Hodgkin lymphoma, AML = acute myeloid leukemia, CLL = chroniclymphocytic leukemia.

The table shows, like Table 4, for selected peptides on which additionaltumor types they were found showing over-presentation (includingspecific presentation) on more than 5% of the measured tumor samples, orpresentation on more than 5% of the measured tumor samples with a ratioof geometric means tumor vs normal tissues being larger than 3.Over-presentation is defined as higher presentation on the tumor sampleas compared to the normal sample with highest presentation. Normaltissues against which over-presentation was tested were: adipose tissue,adrenal gland, blood cells, blood vessel, bone marrow, brain, esophagus,eye, gallbladder, heart, kidney, large intestine, liver, lung, lymphnode, nerve, pancreas, parathyroid gland, peritoneum, pituitary, pleura,salivary gland, skeletal muscle, skin, small intestine, spleen, stomach,thyroid gland, trachea, ureter, urinary bladder.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 5, 8, 14, 19, 22, 29, 30, 31, 35, 37, 46, 60, 69,70, 79, 85, 90, 92, 95, 101, 102, 118, 123, 124, 125, 128, 131, 136,138, 142, 147, 149, 150, 151, 154, 158, 160, 167, 6, 9, 21, 84, 85, 94,96, 99, 111, 113, 114, 116, 129, 134, 152, 159, and 169 for the—in onepreferred embodiment combined—treatment of non-small cell lung cancer(NSCLC).

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 14, 19, 35, 46, 52, 59, 60, 62, 64, 75, 79, 80, 81,90, 95, 102, 110, 114, 124, 125, 128, 129, 131, 136, 138, 143, 144, 145,147, 148, 149, 150, 158, 160, 162, 163, 165, 167, 169, 4, 6, 23, 37, 94,104, and 155 for the—in one preferred embodiment combined—treatment ofsmall cell lung cancer (SCLC).

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 13, 14, 29, 46, 84, 115, 147, 162, 175, 12, 30, 38,75, 95, 99, 111, 130, and 160 for the—in one preferred embodimentcombined—treatment of kidney cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 16, 17, 29, 34, 39, 63, 67, 81, 93, 94, 98, 102, 104,106, 113, 114, 115, 116, 122, 129, 138, 146, 151, 159, 161, 166, 167,169, 172, 11, 14, 19, 70, 71, 83, 87, 99, 112, 123, 126, 132, 152, and160 for the—in one preferred embodiment combined—treatment of braincancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 31, 32, 68, 84, 88, 95, 97, 117, 120, 121, 147, 174,4, 9, 41, 77, 149, and 160 for the—in one preferred embodimentcombined—treatment of stomach cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 5, 28, 29, 30, 31, 37, 40, 41, 60, 85, 90, 92, 93, 94,99, 114, 115, 120, 124, 125, 128, 131, 137, 142, 145, 148, 151, 152,154, 157, 158, 159, 165, 4, 34, 84, 111, 146, 149, and 160 for the—inone preferred embodiment combined—treatment of colon and rectal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 5, 14, 19, 31, 35, 37, 48, 50, 51, 60, 64, 70, 73,80, 85, 86, 96, 99, 100, 101, 102, 103, 104, 111, 114, 115, 116, 120,123, 124, 125, 129, 131, 132, 135, 136, 137, 142, 145, 146, 148, 149,150, 155, 158, 159, 160, 161, 165, 167, 169, 174, and 176 for the—in onepreferred embodiment combined—treatment of liver cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 8, 10, 22, 31, 39, 46, 79, 86, 104, 111, 123, 130,142, 156, and 167 for the—in one preferred embodiment combined—treatmentof pancreatic cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 98, 102, 109, 111, 115, 142, 148, 151, and 167 forthe—in one preferred embodiment combined—treatment of prostate cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 7, 22, 48, 62, 71, 81, 83, 94, 95, 104, 110, 122, 144,145, 147, 149, 150, 152, 154, 159, 160, 161, 171, and 176 for the—in onepreferred embodiment combined—treatment of leukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 3, 4, 21, 29, 30, 32, 52, 57, 65, 67, 84, 93, 99, 102,103, 106, 108, 111, 114, 117, 120, 123, 126, 127, 128, 139, 140, 142,143, 148, 151, and 158 for the—in one preferred embodimentcombined—treatment of AML.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 6, 84, 91, 99, 107, 114, 118, 127, 130, 142, 155,and 158 for the—in one preferred embodiment combined—treatment of CLL.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 4, 8, 9, 16, 22, 26, 31, 35, 37, 41, 59, 77, 79,84, 90, 93, 99, 110, 137, 142, 150, 169, 175, 29, 42, 60, 69, 70, 75,80, 82, 86, 95, 111, 120, 124, 127, 128, 129, 138, 144, 149, 151, 155,158, and 160 for the—in one preferred embodiment combined—treatment ofbreast cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 149, 81, 101, 116, and 124 for the—in one preferredembodiment combined—treatment of Merkel cell carcinoma (MCC).

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 14, 17, 19, 34, 37, 45, 46, 52, 85, 92, 95, 96, 107,113, 114, 115, 118, 124, 131, 138, 146, 149, 150, 155, 157, 158, 161,165, 169, 1, 5, 6, 9, 12, 16, 18, 21, 22, 23, 25, 28, 29, 31, 32, 44,55, 56, 57, 58, 60, 61, 65, 66, 67, 68, 75, 84, 87, 88, 90, 93, 94, 97,99, 106, 111, 116, 117, 120, 121, 123, 127, 128, 129, 130, 132, 133,134, 135, 137, 139, 142, 143, 156, 159, and 160 for the—in one preferredembodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 14, 35, 37, 46, 52, 60, 70, 81, 85, 92, 94, 95,101, 120, 124, 125, 129, 131, 132, 134, 136, 142, 146, 147, 149, 150,154, 157, 158, 160, 162, 165, 167, 3, 47, 57, 84, 89, 99, 111, 138, and148 for the—in one preferred embodiment combined—treatment of ovariancancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 2, 4, 5, 8, 9, 11, 12, 14, 19, 29, 35, 37, 40, 46,50, 51, 52, 57, 58, 62, 63, 67, 70, 75, 77, 79, 85, 91, 92, 94, 95, 106,114, 118, 120, 124, 129, 131, 132, 135, 136, 137, 142, 147, 148, 149,154, 158, 165, 169, 1, 2, 4, 5, 8, 9, 11, 12, 14, 19, 29, 35, 37, 40,46, 50, 51, 52, 57, 58, 62, 63, 67, 70, 75, 77, 79, 85, 91, 92, 94, 95,106, 114, 118, 120, 124, 129, 131, 132, 135, 136, 137, 142, 147, 148,149, 154, 158, 165, and 169 for the—in one preferred embodimentcombined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 3, 4, 20, 29, 37, 41, 42, 59, 60, 64, 82, 88, 91, 93,95, 97, 99, 101, 104, 106, 114, 115, 120, 122, 131, 135, 137, 142, 150,151, 152, 154, 156, 157, 160, 166, 169, 170, 171, 175, 1, 5, 6, 12, 14,15, 19, 30, 31, 38, 46, 52, 57, 65, 67, 70, 71, 74, 84, 86, 89, 92, 94,100, 103, 107, 109, 111, 113, 118, 123, 126, 127, 128, 129, 130, 138,148, 149, 158, and 159 for the—in one preferred embodimentcombined—treatment of urinary bladder cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 41, 85, 94, 116, 120, 130, 143, 162, 165, and 166 forthe—in one preferred embodiment combined—treatment of endometrialcancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 8, 10, 19, 31, 73, 75, 94, 102, 115, 116, 122, 125,131, 149, 4, 21, 22, 30, 46, 50, 69, 70, 80, 90, 95, 96, 103, 111, 120,129, 142, 144, 145, 147, 152, 154, 156, and 160 for the—in one preferredembodiment combined—treatment of gall bladder and bile duct cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 5, 6, 12, 14, 15, 19, 29, 37, 38, 39, 42, 46, 50,56, 57, 60, 70, 74, 82, 84, 91, 95, 101, 103, 104, 106, 110, 111, 118,123, 129, 132, 135, 146, 148, 149, 151, 156, 157, 158, 159, 160, and 161for the—in one preferred embodiment combined—treatment of uterinecancer.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofpancreatic cancer, lung cancer, kidney cancer, brain cancer, stomachcancer, colon or rectal cancer, liver cancer, prostate cancer, leukemia,breast cancer, Merkel cell carcinoma (MCC), melanoma, ovarian cancer,esophageal cancer, urinary bladder cancer, endometrial cancer, gallbladder cancer, and bile duct cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) Class-I or—in an elongatedform, such as a length-variant-MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 161.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 161, preferably containing SEQ IDNo. 1 to SEQ ID No. 79, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are pancreatic cancer, lung cancer,kidney cancer, brain cancer, stomach cancer, colon or rectal cancer,liver cancer, prostate cancer, leukemia, breast cancer, Merkel cellcarcinoma (MCC), melanoma, ovarian cancer, esophageal cancer, urinarybladder cancer, endometrial cancer, gall bladder cancer, bile ductcancer, and preferably pancreatic cancer cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably pancreaticcancer. The marker can be over-presentation of the peptide(s)themselves, or over-expression of the corresponding gene(s). The markersmay also be used to predict the probability of success of a treatment,preferably an immunotherapy, and most preferred an immunotherapytargeting the same target that is identified by the biomarker. Forexample, an antibody or soluble TCR can be used to stain sections of thetumor to detect the presence of a peptide of interest in complex withMHC.

Optionally, the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

AAGAB encodes a protein that interacts with the gamma-adaptin andalpha-adaptin subunits of complexes involved in clathrin-coated vesicletrafficking. Mutations in this gene are associated with type I punctatepalmoplantar keratoderma (RefSeq, 2002). AAGAB is a target of miR-205,which is over-expressed in cervical cancer (Xie et al., 2012).Knock-down of AAGAB leads to increased cell division and proliferation(Pohler et al., 2012).

ACTR2 encodes ARP2 actin-related protein 2 homolog, a major constituentof the ARP2/3 complex. This complex is essential for cell shape andmotility through lamellipodial actin assembly and protrusion (RefSeq,2002). ARP2/3 in complex with other proteins was shown to play acritical role in cancer cell invasion and migration (Nurnberg et al.,2011; Feldner and Brandt, 2002; Frugtniet et al., 2015; Kurisu andTakenawa, 2010; Kirkbride et al., 2011). The ARP2/3 complex withWASP/WAVE protein family members contributes to cell invasion andmigration in breast cancer (Frugtniet et al., 2015). The ARP2/3 complexwith ArgBP2 is endowed with an anti-tumoral function, when the adhesionand migration of pancreatic cancer cells is regulated (Roignot andSoubeyran, 2009).

ADAM9 encodes one member of the ADAM (a disintegrin and metalloproteasedomain) family (member 9). Members of this family take part in thecell-cell and cell-matrix interactions (RefSeq, 2002). ADAM9 genesilencing reduces esophageal squamous cell carcinoma (ESCC) cancerproliferation (Liu et al., 2015b). ADAM9 plays an important role inmelanoma proliferation and invasion (Ebrahimi et al., 2014). ADAM9 wasshown to be up-regulated in osteosarcoma cells, muscle invasive (MI)bladder cancer cells, non-small cell lung cancer, pancreatic cancer,colon cancer, oral squamous cell carcinoma, cervical cancer, prostatecancer, renal cancer, gastric cancer, lymph node cancer, and breastcancer (Shaker et al., 2011; Vincent-Chong et al., 2013; Li et al.,2013; Ebrahimi et al., 2014; Zhang et al., 2014a; Jia et al., 2014;O'Shea et al., 2003; Jiang et al., 2014a; Zubel et al., 2009). ADAM9 hasbeen implicated in lung cancer metastasis to the brain (Sher et al.,2014; Lin et al., 2014a; Shintani et al., 2004). AGAP9 encodes ArfGAPwith GTPase domain, Ankyrin repeat and PH domain 9 and is located onchromosome 10q11.22 (RefSeq, 2002).

AHCY encodes adenosylhomocysteinase. It regulates the intracellularS-adenosylhomocysteine (SAH) concentration thought to be important fortransmethylation reactions (RefSeq, 2002). AHCY down-regulationcontributes to tumorigenesis (Leal et al., 2008). AHCY can promoteapoptosis. It inhibits migration and adhesion of esophageal squamouscell carcinoma cells suggesting a role in carcinogenesis of theesophagus (Li et al., 2014b). AHCY protein expression is up-regulated incolon cancer (Kim et al., 2009; Watanabe et al., 2008; Fan et al.,2011). AHCY may be a potential biomarker in ovarian cancer (Peters etal., 2005).

AK2 encodes adenylate kinase 2. AK2 is localized in the mitochondrialintermembrane space and may play a role in apoptosis (RefSeq, 2002). AK2mediates a novel intrinsic apoptotic pathway that may be involved intumorigenesis (Lee et al., 2007).

ANKLE2 encodes Ankyrin repeat and LEM domain containing 2. ANKLE2 is amember of the LEM family of inner nuclear membrane proteins. The encodedprotein functions as a mitotic regulator through post-mitotic formationof the nuclear envelope (RefSeq, 2002).

ANKRD1 encodes Ankyrin repeat domain-1. It is localized to the nucleusof endothelial cells and is induced by IL-1 and TNF-alpha stimulation.Interactions between this protein and the sarcometric proteinsmyopalladin and titin suggest that it may also be involved in themyofibrillar stretch-sensor system (RefSeq, 2002). The ectopicexpression of ANKRD1 leads to reduced colony formation and to enhancedapoptotic cell death in hepatoma cells (Park et al., 2005). Highexpression of ANKRD1 in ovarian carcinoma is associated with poorsurvival (Lei et al., 2015).

ANLN encodes an actin-binding protein that plays a role in cell growthand migration, and in cytokinesis. ANLN is thought to regulate actincytoskeletal dynamics in podocytes, components of the glomerulus.Mutations in this gene are associated with focal segmentalglomerulosclerosis 8 (RefSeq, 2002). ANLN was found to be highlyexpressed in breast cancer tissues as well as head and neck squamouscell carcinomas. Knock-down of ANLN remarkably inhibited theproliferation rate, colony formation ability and migration of breastcancer cells (Zhou et al., 2015b). ANLN is over-expressed inproliferative gastric tumors, pancreatic carcinoma andhormone-refractory prostate cancers (Pandi et al., 2014; Tamura et al.,2007; Shimizu et al., 2007; Olakowski et al., 2009). ANLN is a biomarkerfor hepatocellular carcinoma (Kim et al., 2013a). ANLN expression is amarker of favorable prognosis in patients with renal cell carcinoma(Ronkainen et al., 2011).

APOL6 encodes apolipoprotein L, 6. APOL6 is a member of theapolipoprotein L gene family. The encoded protein is found in thecytoplasm, where it may affect the movement of lipids or allow thebinding of lipids to organelles (RefSeq, 2002). APOL6 inducesmitochondria-mediated apoptosis in cancer cells (Liu et al., 2005).

ARMC9 (also called KU-MEL-1) encodes an armadillo repeat-containingprotein that was a previously isolated melanoma antigen preferentiallyexpressed in melanocytes. It is associated with Vogt-Koyanagi-Haradadisease (Otani et al., 2006). ARMC9 is strongly expressed in melanomacell lines and tissue samples. Antigens against ARMC9 were detected inthe sera of patients treated against brain, colon and esophageal cancer(Kiniwa et al., 2001).

ASNS encodes asparagine synthetase. The ASNS gene complements a mutationin the temperature-sensitive hamster mutant ts11, which blocksprogression through the G1 phase of the cell cycle at non-permissivetemperature (RefSeq, 2002). ASNS expression is induced by glucosedeprivation and protects pancreatic cancer cells from apoptosis (Cui etal., 2007). ASNS is associated with drug resistance in leukemia anduterine cancer (Lin et al., 2012; Zhang et al., 2013a). Knock-down ofASNS in A375 cells down-regulates the expression levels of CDK4, CDK6,and cyclin D1 and up-regulates the expression of p21 (Li et al., 2015a).Down-regulation of ASNS induces cell cycle arrest and inhibits cellproliferation of breast cancer (Yang et al., 2014a). ASNS is highlyexpressed in gliomas (Panosyan et al., 2014). ASNS is a potentialbiomarker in ovarian cancer (Lorenzi et al., 2006; Lorenzi et al., 2008;Lorenzi and Weinstein, 2009).

ATP5F1 encodes ATP synthase, H+transporting, mitochondrial F0 complex,subunit B1, a subunit of mitochondrial ATP synthase (RefSeq, 2002).ATP5F1 is up-regulated in hepatitis B virus-associated hepatocellularcarcinoma (Lee et al., 2008a).

BMS1 encodes BMS1 ribosome biogenesis factor and is located onchromosome 10q11.21. A similar protein in yeast functions in 35S-rRNAprocessing, which includes a series of cleavage steps critical forformation of 40S ribosomes (RefSeq, 2002; Perez-Fernandez et al., 2011).

BMS1P5 encodes BMS1 ribosome biogenesis factor pseudogene 5 and islocated on chromosome 10q11.22 (RefSeq, 2002).

BRK1 (also called C3orf10 or HSPC300) encodes the smallest subunit ofthe Wave complex and is an important regulator of the Wave/Scar pathwayinvolved in actin cytoskeleton dynamics during embryonic development andcell transformation (Derivery et al., 2008; Escobar et al., 2010). BRK1has oncogenic potential in different cancer types including lung cancerand renal cell carcinomas (Cascon et al., 2007; Cai et al., 2009;Escobar et al., 2010). BRK1 is regulated by the transcription factorsSpl and NRF-1. It is involved in the Wave/Scar pathway following Arp2/3regulation and required for cell proliferation and transformation (Li etal., 2014a; van′t Veer et al., 2006; Escobar et al., 2010; Wang et al.,2013c).

BTBD1 encodes BTB (POZ) domain containing 1. The C-terminus of theprotein binds topoisomerase I. The N-terminus contains proline richregion and a BTB/POZ domain, both of which are typically involved inprotein-protein interactions (RefSeq, 2002).

BUB1B encodes a kinase involved in spindle checkpoint function. Theprotein is localized to the kinetochore and plays a role in theinhibition of the anaphase-promoting complex/cyclosome (APC/C), delayingthe onset of anaphase and ensuring proper chromosome segregation.Impaired spindle checkpoint has been found in many forms of cancer(RefSeq, 2002). BUB1B is a tumor inhibitory protein. BUB1B regulates thespindle assembly checkpoint. BUB1B is inactivated or down-regulated intumors. Mutations in BUB1B are also linked to tumor development (Aylonand Oren, 2011; Fagin, 2002; Malumbres and Barbacid, 2007; Rao et al.,2009). BUB1B is associated with gastric carcinogenesis through oncogenicactivation (Resende et al., 2010). BUB1B mutation is one of the causesfor colorectal cancer (Karess et al., 2013; Grady, 2004).

C11orf70 encodes a protein with uncharacterized function, but is linkedto the binding of a mutated protein that causes amyotrophic lateralsclerosis (Wang et al., 2015i). C11orf70 is down-regulated in testiculargerm cell tumors in comparison to normal testis tissue(Gonzalez-Exposito et al., 2015; Alagaratnam et al., 2009). The geneticregion of C11orf70 displays DNA copy number aberrations in oral squamouscell carcinomas, which is associated with oral cancer-specific mortality(Chen et al., 2015a).

C11orf80 encodes chromosome 11 open reading frame 80 and is located onchromosome 11 q13.2 (RefSeq, 2002).

C11orf198 encodes chromosome 1 open reading frame 198 and is located onchromosome 1q42.2 (RefSeq, 2002).

C20orf24 encodes chromosome 20 open reading frame 24 and is located onchromosome 20q11.23 (RefSeq, 2002). C20orf24 plays an important role inchromosomal instability-related progression from adenoma to carcinoma.C20orf24 is significantly over-expressed in carcinomas compared withadenomas. C20orf24 may serve as a highly specific biomarker forcolorectal cancer (Carvalho et al., 2009).

CAD encodes for trifunctional protein carbamoylphosphate synthetase 2,aspartate transcarbamylase, and dihydroorotase, which catalyzes thefirst three reactions of the pyrimidine biosynthesis pathway (RefSeq,2002). CAD activity is increased in different cancer types, includinghepatomas, sarcomas and kidney adenocarcinomas and is very frequentlyassociated with the amplification of the CAD gene (Smith et al., 1990;Aoki and Weber, 1981; Smith et al., 1997). CAD is a target of differentoncogenes and tumorigenesis regulating pathways like MAPK, mTORC1 andc-Myc (Mac and Farnham, 2000; Graves et al., 2000; Sharma et al., 2014).CAD promotes androgen receptor translocation into the nucleus andstimulates its transcriptional activity in prostate tumor cells. Afterradical prostatectomy a higher CAD mRNA level is associated with localtumor extension and cancer relapse (Morin et al., 2012).

CARM1 encodes coactivator-associated arginine methyltransferase 1. CARM1belongs to the protein arginine methyltransferase (PRMT) family. Theencoded enzyme catalyzes the methylation of guanidine nitrogens ofarginyl residues of proteins. The enzyme is involved in gene expression(RefSeq, 2002). CARM1 has shown to be dysregulated in colorectal andprostate cancer, melanoma and breast cancer. CARM1 is over-expressed notonly in prostate tumors, but also in prostatic intraepithelial neoplasia(PIN). CARM1 is significantly over-expressed in non-small cell lungcarcinomas (NSCLC). CARM1 expression is elevated in adenomas andaberrant in carcinomas during hepatocellular carcinogenesis (Limm etal., 2013; Osada et al., 2013; Elakoum et al., 2014; Baldwin et al.,2014). CARM1 methylates chromatin remodeling factor BAF155 to enhancetumor progression and metastasis (Wang et al., 2014a; Stefansson andEsteller, 2014).

CCNA2 encodes cyclin A2, a member of the highly conserved cyclin family.CCNA2 binds and activates CDC2 or CDK2 kinases, and thus promotes bothcell cycle G1/S and G2/M transitions (RefSeq, 2002). Over-expression ofCCNA2 inhibits the proliferation of hepatocellular carcinoma cells.Over-expression of CCNA2 in endometrial adenocarcinoma cells decreasescell growth and increases apoptosis. CCNA2 expression in melanoma cellsreduces tumor growth and metastasis and concomitantly increasesapoptosis in tumors (Lau, 2011). CCNA2 can promote cancer cellproliferation, invasion, adhesion, differentiation, survival andmetastasis. It plays an important role in angiogenesis and extracellularmatrix production. CCNA2 promotes tumor growth and increases tumorvascularization when over-expressed in gastric adenocarcinoma cells.

Silencing of CCNA2 expression decreases tumor growth in pancreaticcancer cells. CCNA2 can promote the proliferation of prostate cancercells (Lau, 2011; Chen and Du, 2007). CCNA2 over-expression inducesepithelial-mesenchymal transition, leading to laryngeal tumor invasionand metastasis (Liu et al., 2015e). CCNA2 is dysregulated in colorectalcancer (Chang et al., 2014). CCNA2 is over-expressed in prostate cancer,gliomas, pancreatic cancer, and breast cancer. CCNA2 is associated withincreased aggressiveness, vascularization, and estrogen independence inbreast cancer, suggesting a major role of CCNA2 in breast cancerprogression (Zuo et al., 2010).

CCND1 encodes cyclin D1. It belongs to the highly conserved cyclinfamily, whose members are characterized by a dramatic periodicity inprotein abundance throughout the cell cycle. Mutations, amplificationsand over-expression of CCND1, which alters cell cycle progression, areobserved frequently in a variety of tumors and may contribute totumorigenesis (RefSeq, 2002). CCND1 is amplified and over-expressed incases of lymph node metastasis in oral squamous cell carcinoma,gastrointestinal stromal tumor, non-small cell lung cancer, pituitarytumors and breast cancer (Noorlag et al., 2015; Dworakowska, 2005;Gautschi et al., 2007; Lambros et al., 2007; Yang et al., 2008; Yu andMelmed, 2001). CCND1 is over-expressed in mantle cell lymphoma,pancreatic neuroendocrine tumors, parathyroid adenoma, and Ewing sarcoma(Navarro et al., 2011; Sander, 2011; Capurso et al., 2012; Delas et al.,2013; Setoodeh et al., 2013; Sanchez et al., 2008; Westin et al., 2009).CCND1 can increase colorectal cancer risk (Yang et al., 2012b; Andersenet al., 2013). CCND1 genetic alterations can cause bladder cancer (Zhanget al., 2003; Baffa et al., 2006).

CCT3 encodes chaperonin containing TCP1, subunit 3 (gamma), a molecularchaperone (RefSeq, 2002). CCT3 is elevated in hepatocellular carcinoma(Midorikawa et al., 2002; Skawran et al., 2008). CCT3 is a potentiallynovel biomarker for ovarian cancer (Peters et al., 2005).

CCT4 encodes chaperonin containing TCP1, subunit 4. CCT4 assists thefolding of newly translated polypeptide substrates through multiplerounds of ATP-driven release and rebinding of partially foldedintermediate forms (RefSeq, 2002). CCT4 deregulation causes esophagealsquamous cell carcinoma and lung adenocarcinoma (Wang et al., 2015j;Tano et al., 2010). CCT4 is upregulated in gastric cancers (Malta-Vacaset al., 2009).

CDC27 encodes cell division cycle 27. The protein encoded by this geneis a component of the anaphase-promoting complex (APC). The protein maybe involved in controlling the timing of mitosis (RefSeq, 2002). CDC27confers increased radio-resistance of triple negative breast cancercells and squamous cell cervix carcinoma, when it is down-regulated(Rajkumar et al., 2005; Ren et al., 2015). CDC27 plays a crucial role inthe progression of hepatocellular carcinoma, and also correlates withpoor prognosis in esophageal squamous cell carcinoma and pancreaticcancer (Ahn et al., 2014; Wang et al., 2015h). CDC27 polymorphisms maycontribute to the susceptibility of breast cancer through influencingthe mitotic progression of cells (Guo et al., 2015). CDC27 mutation isinvolved in prostate cancer (Lindberg et al., 2013). CDC27 mutation anddown-regulation is involved in several breast and colon carcinoma celllines (Fan et al., 2004; Roy et al., 2010; Pawar et al., 2010).

CDK12 encodes cyclin dependent kinase 12 and is located on chromosome17q12 (RefSeq, 2002). CDK12 mutations were identified in a variety oftumors, including ovarian, breast, prostate, and intestinal tumors(Vrabel et al., 2014).

CDK13 encodes cyclin dependent kinase 13, a member of the cyclindependent serine/threonine protein kinase family. Members of this familyare known for their essential roles as master switches in cell cyclecontrol. They may play a role in mRNA processing and may be involved inregulation of hematopoiesis (RefSeq, 2002). CDK13 is associated withpancreatic cancer and skin cancer (Ansari et al., 2015; Nelson et al.,1999; Chandramouli et al., 2007). CDK13 is amplified in hepatocellularcarcinoma (Kim et al., 2012b).

CDK2 encodes cyclin dependent kinase 2, a serine/threonine proteinkinase that participates in cell cycle regulation. Activity of thisprotein is especially critical during the G1 to S phase transition(RefSeq, 2002). CDK2 over-expression indicates the abnormal regulationof cell cycle, which would be directly related to hyper-proliferation incancer cells (Chohan et al., 2015). CDK2 is associated with leukemia,colorectal carcinoma, melanoma, human papillomavirus-associated cervicalneoplasia, lung cancer, breast cancer and prostate cancer (Foster etal., 2001; Zajac-Kaye, 2001; Raso et al., 2013; He et al., 2013;Duensing and Munger, 2002; Hu and Zuckerman, 2014; Agarwal, 2000).

CDK2 is highly-expressed in mantle cell lymphoma (Rummel et al., 2004).

CDK5RAP3 encodes CDK5 regulatory subunit associated protein 3. CDK5RAP3plays a role in signaling pathways governing transcriptional regulationand cell cycle progression.

It may have a function in tumorigenesis and metastasis (RefSeq, 2002).CDK5RAP3 is over-expressed in hepatocellular carcinoma and promotesmetastasis (Mak et al., 2011; Mak et al., 2012).

CDK7 encodes cyclin dependent kinase 7, a member of the cyclin dependentprotein kinase family. It is an essential component of the transcriptionfactor TFIIH, which is involved in transcription initiation and DNArepair. This protein is thought to serve as a direct link between theregulation of transcription and the cell cycle (RefSeq, 2002). CDK7genetic polymorphisms predispose individuals to breast cancer bygene-environment or gene-gene interactions (Yoo and Kang, 2003). CDK7 isassociated with an increased risk for pancreatic cancer (Efthimiou etal., 2001). CDK7 has been associated with breast cancer (Cance and Liu,1995).

CDK9 encodes cyclin dependent kinase 9, a member of the cyclin dependentprotein kinase family. This protein forms a complex with and isregulated by its regulatory subunit cyclin T or cyclin K (RefSeq, 2002).CDK9 appears to be involved in the differentiation program of severalcell types, such as muscle cells, monocytes and neurons. CDK9 seems tohave an anti-apoptotic function in monocytes. Involvement of CDK9 inseveral physiological processes in the cell may lead to the onset ofcancer (De and Giordano, 2002).

CELSR3 encodes cadherin, EGF LAG seven-pass G-type receptor 3. Theencoded protein may be involved in the regulation of contact dependentneurite growth and may play a role in tumor formation (RefSeq, 2002).Microarray screening revealed that CELSR3 hyper-methylated in primaryoral squamous cell carcinoma compared to normal oral mucosa (Khor etal., 2014). CELSR3 is associated with ovarian cancer and brain tumors(Asad et al., 2014; Katoh and Katoh, 2007). CELSR3 is up-regulated inpancreatic and hepatic tumor stellate cells (Erkan et al., 2010).

CEP97 encodes centrosomal protein 97 kDa and is located on chromosome3q12.3 (RefSeq, 2002). CEP97 is associated with breast cancer (Rappa etal., 2014).

CFL1 encodes cofilin 1. It is involved in the translocation of theactin-cofilin complex from cytoplasm to nucleus (RefSeq, 2002). CFL1mutation is associated with multiple endocrine neoplasia type 4 andglioblastoma multiforme (Solomon et al., 2008; Georgitsi, 2010). CFL1 isover-expressed in lymphoma, leukemia, neuroblastoma, ovarian, prostate,breast and lung cancers and mesothelioma (Rana et al., 2008). CFL1 isdown-regulated in testicular germ cell tumors (von Eyben, 2004).

CHD3 encodes chromodomain helicase DNA binding protein 3. The protein isone of the components of a histone deactelylase complex referred to asthe Mi-2/NuRD complex which participates in the remodeling of chromatinby deacetylating histones (RefSeq, 2002). CHD3 is up-regulated inpancreatic intraepithelial neoplasia and pancreatic carcinoma (Wang etal., 2011). CHD3 mutation is associated with gastric and colorectalcancer (Kim et al., 2011a). CHD3 is over-expressed in acute myeloidleukemia (Camos et al., 2006).

CHD4 encodes chromodomain helicase DNA binding protein 4. It representsthe main component of the nucleosome remodeling and deacetylase complexand plays an important role in epigenetic transcriptional repression.Somatic mutations in this gene are associated with serous endometrialtumors (RefSeq, 2002). CHD4 is a novel therapeutic target for acutemyeloid leukemia (Sperlazza et al., 2015). CHD4 epigenetically controlsgene regulation and DNA damage responses in EpCAM+liver cancer stemcells (Nio et al., 2015). CHD4 modulates therapeutic response in BRCA2mutant cancer cells (Guillemette et al., 2015). CHD4 is associated withglioblastoma and colon cancer (Cai et al., 2014; Chudnovsky et al.,2014).

CHD5 encodes chromodomain helicase DNA binding protein 5. CHD5 is apotential tumor suppressor that may play a role in the development ofneuroblastoma (RefSeq, 2002).

CHD5 functions as a tumor suppressor gene in gliomas and a variety ofother tumor types, including breast, colon, lung, ovary, and prostatecancer (Kolla et al., 2014).

CIRH1A (also called Cirhin) encodes cirrhosis autosomal recessive 1 A, aWD40-repeat-containing protein localized in the nucleolus. It causesNorth American Indian childhood cirrhosis (NAIC) (RefSeq, 2002). CIRH1Acan up-regulate a canonical NF-kappaB element and might participate inthe regulation of other genes containing NF-kappaB elements. Thissuggests that CIRH1A can influence the cancer-related NF-kappaB pathway(Yu et al., 2009).

COL1A1 encodes collagen, type 1, alpha 1. Type 1 is a fibril formingcollagen found in most connective tissues and is abundant in bone,cornea, dermis, and tendon. Reciprocal translocations betweenchromosomes 17 and 22, where this gene and the gene for platelet derivedgrowth factor beta are located, are associated with a particular type ofskin tumor called dermatofibrosarcoma protuberans, resulting fromunregulated expression of the growth factor (RefSeq, 2002). COL1A1 isdifferentially expressed in gastric cancer (Yasui et al., 2004). COL1A1is associated with pigmented dermatofibrosarcoma protuberans (Zhang etal., 2013c).

COL1A2 encodes collagen, type 1, alpha 2. Type 1 is a fibril formingcollagen found in most connective tissues and is abundant in bone,cornea, dermis and tendon (RefSeq, 2002). COL1A2 is associated withgastric cancer (Yasui et al., 2004; Yasui et al., 2005).

COL6A1 encodes collagen, type 6, alpha 1. Collagen VI is a majorstructural component of microfibrils. Mutations in the genes that codefor the collagen VI subunits result in the autosomal dominant disorderBethlem myopathy (RefSeq, 2002). COL6A1 is up-regulated in the reactivestroma of castration-resistant prostate cancer and promotes tumor growth(Zhu et al., 2015c). COL6A1 is over-expressed in CD166− pancreaticcancer cells that show stronger invasive and migratory activities thanthose of CD166+ cancer cells (Fujiwara et al., 2014). COL6A1 is highlyexpressed in bone metastasis (Blanco et al., 2012). COL6A1 was found tobe up-regulated in cervical and ovarian cancer (Zhao et al., 2011;Parker et al., 2009). COL6A1 is differentially expressed in astrocytomasand glioblastomas (Fujita et al., 2008).

COL6A3 encodes collagen, type VI, alpha 3, one of the three alpha chainsof type VI collagen, a beaded filament collagen found in most connectivetissues, and important in organizing matrix components (RefSeq, 2002).COL6A3 encodes the alpha-3 chain of type VI collagen, a beaded filamentcollagen found in most connective tissues, playing an important role inthe organization of matrix components (RefSeq, 2002). COL6A3 isalternatively spliced in colon, bladder and prostate cancer. The longisoform of COL6A3 is expressed almost exclusively in cancer samples andcould potentially serve as a new cancer marker (Thorsen et al., 2008).COL6A3 is highly expressed in pancreatic ductal adenocarcinoma tissueand undergoes tumor-specific alternative splicing (Kang et al., 2014).COL6A3 has been demonstrated to correlate with high-grade ovarian cancerand contributes to cisplatin resistance. COL6A3 was observed to befrequently over-expressed in gastric cancer tissues (Xie et al., 2014).COL6A3 mutation(s) significantly predicted a better overall survival inpatients with colorectal carcinoma independent of tumor differentiationand TNM staging (Yu et al., 2015b). COL6A3 expression was reported to beincreased in pancreatic cancer, colon cancer, gastric cancer,mucoepidermoid carcinomas and ovarian cancer. Cancer associatedtranscript variants including exons 3, 4 and 6 were detected in coloncancer, bladder cancer, prostate cancer and pancreatic cancer (Arafat etal., 2011; Smith et al., 2009; Yang et al., 2007; Xie et al., 2014;Leivo et al., 2005; Sherman-Baust et al., 2003; Gardina et al., 2006;Thorsen et al., 2008). In ovarian cancer COL6A3 levels correlated withhigher tumor grade and in pancreatic cancer COL6A3 was shown torepresent a suitable diagnostic serum biomarker (Sherman-Baust et al.,2003; Kang et al., 2014).

COPG1 (also called COPG) encodes for the gamma subunit of the coatomerprotein complex (COPI) that mediates retrograde transport from the Golgiback to the ER and intra-Golgi transport. COPG1 binds to ARF-GAP (Waterset al., 1991; Watson et al., 2004). COPG1 correlates with the age of thepatients as well as a higher grade of malignancy and the grade ofgliosarcomas (Coppola et al., 2014). COPG1 was found abundantlyexpressed in lung cancer and lung cancer-related endothelial cells (Parket al., 2008).

CREB3L1 encodes cAMP responsive element binding protein 3-like 1. Inresponse to ER stress, CREB3L1 is cleaved and the released cytoplasmictranscription factor domain translocates to the nucleus. There itactivates the transcription of target genes by binding to box-B elements(RefSeq, 2002). CREB3L1 mutations are frequently found in sclerosingepithelioid fibrosarcoma (SEF) (Prieto-Granada et al., 2015). CREB3L1 isinduced by ER stress in human glioma cell lines and contributes to theunfolded protein response, extracellular matrix production and cellmigration (Vellanki et al., 2013). CREB3L1 is epigenetically silenced inbladder cancer, facilitating tumor cell spreading and migration (Rose etal., 2014). CREB3L1 plays an important role in suppressing tumorigenesisin breast cancer. Loss of expression is required for the development ofa metastatic phenotype (Mellor et al., 2013).

CSTF1 encodes cleavage stimulation factor, 3′ pre-RNA, subunit 1, 50kDa. It is involved in the polyadenylation and 3′ end cleavage ofpre-mRNAs (RefSeq, 2002). CSTF1 variation was found to be associatedwith breast cancer risk in BRCA2 mutation carriers (Blanco et al.,2015).

CTHRC1 encodes collagen triple helix repeat containing 1. CTHRC1 mayplay a role in the cellular response to arterial injury throughinvolvement in vascular remodeling. Mutations at this locus have beenassociated with Barrett esophagus and esophageal adenocarcinoma (RefSeq,2002). CTHRC1 shows increased expression in gastric cancer and ductalcarcinoma of the breast (Kim et al., 2013b; Yu et al., 2015a; Song etal., 2015). CTHRC1 is up-regulated in colorectal cancer (Yan et al.,2015a; Yan et al., 2015b). CTHRC1 expression is highly correlated withhepatocellular carcinoma progression in patients infected with hepatitisB virus. CTHRC1 enhances colony formation, migration and invasion ofhepatoma cells (Tameda et al., 2014; Zhang et al., 2015b). CTHRC1 isover-expressed in non-small cell lung cancer. Over-expression isassociated with tumor aggressiveness and poor prognosis (Ke et al.,2014b). CTHRC1 is up-regulated in esophageal squamous cell carcinoma andBarrett's adenocarcinoma (Timme et al., 2014). CTHRC1 promotes celladhesion and survival in melanoma (Ip et al., 2011).

CXCL5 encodes chemokines C-X-C motif ligand 5. This protein is proposedto bind the G-protein coupled receptor chemokine C-X-C motif receptor 2to recruit neutrophils, to promote angiogenesis and to remodelconnective tissues. This protein is thought to play a role in cancercell proliferation, migration, and invasion (RefSeq, 2002). CXCL5 playsa crucial role in survival, growth and metastasis of renal cellcarcinoma (Parihar and Tunuguntla, 2014). CXCL5 is involved in thetransition of chronic inflammation to esophageal and gastric cancer(Verbeke et al., 2012). CXCL5 is associated with acute myelogenousleukemia (Kittang et al., 2010).

DCBLD2 encodes discoidin, CUB and LCCL domain-containing protein 2 alsoreferred to as endothelial and smooth muscle cell-derivedneuropilin-like protein, a transmembrane co-receptor protein (RefSeq,2002). DCBLD2 is up-regulated in glioblastomas and head and neck cancers(HNCs) and is required for EGFR-stimulated tumorigenesis (Feng et al.,2014). Furthermore, DCBLD2 is up-regulated in highly metastatic lungcancer sublines and tissue samples (Koshikawa et al., 2002). Incontrast, the expression of DCBLD2 is silenced by hypermethylation ofits promoter in gastric cancer (Kim et al., 2008).

DDX43 encodes DEAD (Asp-Glu-Ala-Asp) box polypeptide 43. DDX43 is an ATPdependent RNA helicase and displays tumor specific expression (RefSeq,2002). DDX43 is over-expressed in uveal melanoma cells and in acute andchronic myeloid leukemia (Chen et al., 2011a; Lin et al., 2014b;Ambrosini et al., 2014). DDX43 is a biomarker for breast cancerprognosis (Wiese and Pajeva, 2014). DDX43 is expressed on glioma celllines (Akiyama et al., 2014).

DDX53 encodes DEAD (Asp-Glu-Ala-Asp) box polypeptide 53. DDX53 containsseveral domains found in members of the DEAD box helicase protein family(RefSeq, 2002).

Cancer/testis antigen DDX53 exerts negative regulation on p53 expressionthrough HDAC2 and confers resistance to anti-cancer drugs (Kim et al.,2010b). miR-200b and cancer/testis antigen DDX53 form a feedback loop toregulate the invasion and tumorigenic and angiogenic responses of acancer cell line to microtubule-targeting drugs (Kim et al., 2013c).miR-217 and DDX53 form a feedback loop to regulate the response toanti-cancer drugs through EGFR and HER2 (Kim et al., 2016). DDX53 is oneof several genes with an abnormal DNA hypo-methylation status in uterineleiomyoma (Maekawa et al., 2011). In cell lines derived from 21 B-celland 4 T-cell malignancies, a broad mRNA expression profile was observedfor DDX53 (Liggins et al., 2010).

DNAJC7 encodes DnaJ (Hsp40) homolog, subfamily C, member 7, a member ofthe DNAJ heat shock protein (HSP) 40 family of proteins. This proteinbinds the chaperone proteins HSP70 and HSP90 in an ATP dependent mannerand may function as a co-chaperone (RefSeq, 2002). DNAJC7 enhances p53stability and activity through blocking the complex formation betweenp53 and MDM2 (Kubo et al., 2013).

DPP9 encodes dipeptidyl peptidase 9. DPP9 appears to be involved in theregulation of the activity of its substrates and has been linked to avariety of diseases including type 2 diabetes, obesity and cancer(RefSeq, 2002). DPP9 plays a potential role in breast and ovarian cancer(Wilson and Abbott, 2012). DPP9 plays an important signaling role in theregulation of cell survival and proliferation pathways (Yao et al.,2011). DPP9 mRNA levels are elevated in testicular tumors (Yu et al.,2010). DPP9 is over-expressed in meningiomas (Stremenova et al., 2010).

DPYD (also known as DPD) encodes dihydropyrimidine dehydrogenase, apyrimidine catabolic enzyme and the initial and rate-limiting factor inthe pathway of uracil and thymidine catabolism. Mutations in this generesult in dihydropyrimidine dehydrogenase deficiency, an error inpyrimidine metabolism associated with thymine-uraciluria and anincreased risk of toxicity in cancer patients receiving 5-fluorouracilchemotherapy (RefSeq, 2002). The DPYD expression level can be used as apredictive factor for the efficacy of chemotherapy in gastric cancer(Wan et al., 2016). Statistically significant associations were foundbetween DPYD variants and increased incidence of grade 3 or greaterfluorouracil-related adverse events in patients treated with adjuvantfluorouracil-based combination chemotherapy (Cavalcante et al., 2015;Lee et al., 2016; Boige et al., 2016). There is a correlation betweenDPYD polymorphism and KRAS wild type expression in colorectal cancer(Kleist et al., 2015). The up-regulation of DPYD gene expression leadsto fluoropyrimidine toxicity in colorectal cancer (Chai et al., 2015;Falvella et al., 2015; van Staveren et al., 2015; Nakamura et al., 2015;Chen et al., 2015c; Hu et al., 2015b). Polymorphic expression of DPYDmay be important in determining the treatment response in patients withhead and neck cancer, pancreatic cancer, esophageal squamous cellcarcinoma, digestive cancer, gastric cancer, hepatocellular carcinoma,and colorectal cancer (Kim et al., 2015; Toffoli et al., 2015; Ishizukaet al., 2015; Baba et al., 2015; Launay et al., 2016; Kikuchi et al.,2015; Li et al., 2016; Shimamoto et al., 2016; Bai et al., 2015; Dhawanet al., 2016).

DROSHA, one of the two critical enzymes in microRNA biosynthesis, isover-expressed in a number of cancers including gastrointestinal tumors,breast cancer and cervical cancer and appears to enhance proliferation,colony formation and migration of tumor cells (Avery-Kiejda et al.,2014; Havens et al., 2014; Zhou et al., 2013). DSEL encodes dermatansulfate epimerase-like and is located on chromosome 18q22.1 (RefSeq,2002). DSE is an important paralog of DSEL. DSE is an immunogenic targetfor immunotherapy of hepatocellular carcinoma and colorectal carcinoma(Mizukoshi et al., 2011; Sasatomi et al., 2002).

DST (also known as bullous pemphigoid antigen I (BPAG1)) encodesdystonin, a member of the plakin protein family of adhesion junctionplaque proteins. The full-length isoform is not defined, however, thereare several isoforms expressed in neural and muscle tissue or inepithelial tissue, anchoring either neural intermediate filaments to theactin cytoskeleton or keratin-containing intermediate filaments tohemidesmosomes (RefSeq, 2002; Bouameur et al., 2014; Li et al., 2007).DST may be related to breast cancer metastasis (Sun et al., 2006).Autoantibodies against DST can be found in lymphocytic leukemia andfollicular lymphomas (Aisa et al., 2005; Taintor et al., 2007). DST isup-regulated in 5-8F cells (high tumorigenic and metastatic ability) incomparison to 6-10B cells (tumorigenic, but lacking metastatic ability)in nasopharyngeal carcinoma (Fang et al., 2005). DST is highly expressedin head and neck squamous cell carcinoma (Lin et al., 2004). There areautoantibodies against DST in paraneoplastic pemphigus which isassociated with neoplasms (Yong and Tey, 2013; Wang et al., 2005; Preiszand Karpati, 2007; Zhu and Zhang, 2007). DST expression in prostatecancer is strongly inverse correlated with progression (Vanaja et al.,2003). Anti-DST autoantibodies are a promising marker for the diagnosisof melanoma (Shimbo et al., 2010). DST can be found in the urine ofcachectic cancer patients (Skipworth et al., 2010). DST isdifferentially expressed in adenocarcinomas and squamous cell carcinomasof the lung (McDoniels-Silvers et al., 2002). DST is distinctlyup-regulated with the onset of invasive cell growth (Herold-Mende etal., 2001).

DYNC1H1 encodes the dynein heavy chain 1, a subunit of the main motorprotein for retrograde transport along microtubules. A whole exomesequencing study uncovered somatic mutations within the DYNC1H1 gene inpatients with intra-ductal papillary mucinous neoplasm of the pancreas(Furukawa et al., 2011).

EIF3C encodes eukaryotic translation initiation factor 3, subunit C andis located on chromosome 16p11.2 (RefSeq, 2002). EIF3C is over-expressedand promotes cell proliferation in human U-87 MG cells (Hao et al.,2015). EIF3C is highly expressed in colon cancer (Song et al., 2013).EIF3C mRNA is over-expressed in testicular seminomas (Rothe et al.,2000).

EIF3CL encodes eukaryotic translation initiation factor 3, subunitC-like. It is located on chromosome 16p11.2 (RefSeq, 2002).

EIF3E encodes eukaryotic translation initiation factor 3, subunit E andis located on chromosome 8q22-q23 (RefSeq, 2002). EIF3E might play arole in the carcinogenesis of oral squamous cell carcinoma (Yong et al.,2014). EIF3E is essential for proliferation and survival of glioblastomacells (Sesen et al., 2014). EIF3E has an oncogenic role in breast cancerprogression. Decreased EIF3E expression causes epithelial to mesenchymaltransition in breast epithelial cells (Gillis and Lewis, 2013; Grzmil etal., 2010). EIF3E expression level is significantly increased in bladdercancer (Chen et al., 2011b). EIF3E is involved in non-small lungcarcinoma (Marchetti et al., 2001).

EXT2 encodes exostosin glycosyltransferase 2, one of twoglycosyltransferases involved in the chain elongation step of heparinsulfate biosynthesis. Mutations in his gene cause the type II form ofmultiple exostoses (RefSeq, 2002). EXT2 mutation plays a role inchondrosarcoma (Samuel et al., 2014). EXT2 mutation induces multipleosteochondroma syndrome (Jochmann et al., 2014). EXT2 mutation causeshereditary multiple exostoses, leading to heparan sulfate deficiency(Huegel et al., 2013).

F2R (also known as PAR1) encodes coagulation factor II thrombinreceptor, a transmembrane receptor involved in the regulation ofthrombotic response (RefSeq, 2002). F2R binds to the pleckstrin homology(PH) domain of Etk/Bmx. A F2R mutant, which is unable to bind the PHdomain, reduces mammary tumors and extravillous trophoblast invasion(Kancharla et al., 2015). F2R is thought to promote cancer invasion andmetastasis by facilitating tumor cell migration, angiogenesis, andinteractions with host vascular cells (Wojtukiewicz et al., 2015).Down-regulation of F2R leads to cancer cell death (Burns and Thevenin,2015). Polymorphisms in F2R are associated with acute injury in rectalcancer patients (Zhang et al., 2015a). F2R is correlated with poorprognosis specifically in ER-negative breast cancer patients (Lidfeldtet al., 2015). F2R-deficient mice show reduced colonic adenocarcinomagrowth (Adams et al., 2015). Matrix metalloproteinase (MMP)-1 activatesF2R to induce angiogenesis (Fan et al., 2015). F2R is involved in PTENdown-regulation in lung cancer (Xu et al., 2015). F2R activation inducesthe Hippo-YAP pathway which is correlated with epithelial mesenchymaltransition (Jia et al., 2015; Owens et al., 2015; Yang et al., 2015a;Fujimoto et al., 2015). Inhibition of F2R activation reduces cancer cellmigration and invasion in HER-2 negative breast cancer, hepatocellularcarcinoma and gastric cancer (Mussbach et al., 2015; Wang et al., 2015g;Gonda et al., 2015).

FADS2 encodes fatty acid desaturase 2, a member of the fatty aciddesaturase gene family. Desaturase enzymes regulate unsaturation offatty acids through the introduction of double bonds between definedcarbons of the fatty acyl chain (RefSeq, 2002). FADS2 is up-regulated inhepatocellular carcinoma (Muir et al., 2013). FADS2 activity isincreased in breast cancer tissue (Pender-Cudlip et al., 2013). FADS2expression is associated with aggressiveness of breast cancer (Lane etal., 2003). FADS2 inhibition impedes intestinal tumorigenesis(Hansen-Petrik et al., 2002).

FADS3 encodes fatty acid desaturase 3. Desaturase enzymes regulateunsaturation of fatty acids through the introduction of double bondsbetween defined carbons of the fatty acyl chain (RefSeq, 2002).

FAM83D encodes family with sequence similarity 83, member D and islocated on chromosome 20q11.23 (RefSeq, 2002). Up-regulation of FAM83Daffects the proliferation and invasion of hepatocellular carcinoma cells(Wang et al., 2015a; Liao et al., 2015). FAM83D is significantlyelevated in breast cancer cell lines and in primary human breast cancers(Wang et al., 2013e).

FN1 encodes fibronectin 1, a glycoprotein present in a soluble dimericform in plasma, and in a dimeric or a multimeric form at the cellsurface and in extracellular matrix. It is involved in cell adhesion andmigration processes including embryogenesis, wound healing, bloodcoagulation, host defense, and metastasis (RefSeq, 2002). FN1 is animportant tumor-associated angiogenesis targeting agent (Sollini et al.,2015). FN1 is one of several biomarkers for pancreatic cancer (Ansari etal., 2014). FN1 is one of many factors responsible for endocrineresistance in breast cancer. FN1 is significantly deregulated andpromotes tumor progression and metastatic spread in breast cancer(Oskarsson, 2013; Zheng et al., 2014). It is a biomarker ofepithelial-mesenchymal transition in squamous cell carcinoma (Scanlon etal., 2013). FN1 plays an important role in multiple myeloma (Neri andBahlis, 2012).

FUCA2, secreted human a-I-fucosidase 2, was identified to be the keyenzyme responsible for the transfer of I-fucose. The hydrolytic enzymewas found to be essential for H. pylori adhesion to human gastric cancercells and shows great potential as a diagnostic marker and a target fortherapeutic treatment H. pylori associated gastric cancer (Liu et al.,2009).

GCG encodes glucagon. It is a pancreatic hormone that counteracts theglucose lowering action of insulin by stimulating glycogenolysis andgluconeogenesis. It is a ligand for a specific G-protein linked receptorwhose signaling pathway controls cell proliferation (RefSeq, 2002). GCGreceptor imaging seems to be a potential tool to evaluate pancreaticbeta cell mass. It might also become a target for imaging other tumorssuch as gastrinoma, pheochromocytoma and medullary thyroid cancer(Hubalewska-Dydejczyk et al., 2015). GCG plays a key role in coloncarcinogenesis (Kannen et al., 2013). GCG is an emerging tracer forneuroendocrine tumors (Reubi and Maecke, 2008).

GFPT2 encodes glutamine fructose 6 phosphate transaminase 2 and islocated on chromosome 5q34-q35 (RefSeq, 2002). GFPT2 plays an importantrole in breast cancer and lymphocytic leukemia (Kuang et al., 2008;Simpson et al., 2012).

GPN1 encodes GPN loop GTPase 1 and is located on chromosome 2p23.3(RefSeq, 2002). GPN1 is a cytoplasmic GTPase involved in nuclearlocalization of the DNA repair gene XPA, a critical factor controllingnucleotide excision repair signaling pathways (Nitta et al., 2000).

GRIK2 encodes glutamate receptor, ionotropic, kainite 2. Mutations inthis gene have been associated with autosomal recessive mentalretardation (RefSeq, 2002). TRMT11-GRIK2 is one of several fusiontranscripts found in prostate cancer and is associated with tumoraggressiveness (Yu et al., 2014). GRIK2 SNPs are associated withincreased risk or susceptibility to oral cancer (Bhatnagar et al.,2012). GRIK2 is a potential biomarker for lung cancer (Rauch et al.,2012). GRIK2 inactivation by chromosomal deletion may contribute to theonset of T-cell lymphomas. GRIK2 inactivation plays a role in gastriccarcinogenesis (Resende et al., 2011; Lopez-Nieva et al., 2012).

GRIK3 encodes glutamate receptor, ionotropic, kainite 3. It belongs to afamily of glutamate receptors, which are the predominant excitatoryneurotransmitter receptors in the mammalian brain and are activated in avariety of normal neurophysiologic processes (RefSeq, 2002). GRIK3 isassociated with lung adenocarcinoma (methylation, functionalmodifications), pediatric central nervous system tumors, lymphocyticleukemia, and neuroblastoma (Pradhan et al., 2013). GRIK3 isdifferentially expressed in several pediatric tumors of the centralnervous system (Brocke et al., 2010).

GSK3B encodes glycogen synthase kinase 3 beta. It is involved in energymetabolism, neuronal cell development, and body pattern formation(RefSeq, 2002). Aberrant regulation of GSK3B has been shown to promotecell growth in some cancers, while suppressing it in others, and mayplay an important role in esophageal cancer (Gao et al., 2014b). GSK3Bis dysregulated in glioblastoma multiforme (Atkins et al., 2013).Deregulated GSK3B promotes gastrointestinal, pancreatic, and livercancers (Miyashita et al., 2009).

HLA-A encodes the major histocompatibility complex class 1 A that playsa central role in the immune system by presenting peptides derived fromthe endoplasmic reticulum lumen (RefSeq, 2002). The loss of HLA-Aantigens is a common feature in human tumors. Decrease in the percentageof HLA-A, HLA-B, and HLA-C-positive cells, selective loss of particularantigens and total loss of class 1 molecule expression is documented inmelanomas, carcinomas, lymphomas, neuroblastomas and acute leukemias(Garrido and Ruiz-Cabello, 1991; Salerno et al., 1990). HLA-A expressionis predominantly regulated by the MAPK pathway in gastric and esophagealcancer and in part influenced by the Akt pathway with a strong inversecorrelation between p-Erk expression and HLA class 1 expression inclinical tumor samples (Mimura et al., 2013).

HNRNPU (also called SAF-A) encodes the heterogeneous nuclearribonucleoprotein U that belongs to the RNA binding subfamily ofheterogeneous nuclear riboproteins (hnRNPs), which is associated withpre-mRNA processing and other aspects of mRNA metabolism and transportin the nucleus. HNRNPU is thought to be involved in the packaging ofhnRNA into large ribonucleoprotein complexes (RefSeq, 2002).Up-regulation of the miR-193a-3p that inhibits the metastasis of lungcancer cells down-regulates the expression of HNRNPU (Deng et al.,2015b). The long non-coding RNA H19 can—via association with theHNRNPU/PCAF/RNAPol II protein complex—activate the miR-200 pathway, thuscontributing to mesenchymal-to-epithelial cell transition and to thesuppression of tumor metastasis in hepatocellular carcinoma (Zhang etal., 2013d). HNRNPU interacts with SOX2, a key gene for maintaining thestemness of embryonic and adult stem cells that appears to bere-activated in several human cancers (Fang et al., 2011).

HSPA2 encodes the testis specific heat-shock protein 70-2, essential forthe growth of spermatocytes and cancer cells. Different studies suggestan important role of HSPA2 in disease progression of cervical cancer,renal cell carcinoma and bladder cancer. Polymorphisms within the geneare associated with the development of gastric cancer (Ferrer-Ferrer etal., 2013; Garg et al., 2010a; Garg et al., 2010b; Singh and Suri,2014). HSPA8 was shown to be over-expressed in esophageal squamous cellcarcinoma. High expression levels of HSPA8 in esophageal cancer cellscounter-acted oxidative stress-induced apoptosis of these cells invitro. Furthermore, HSPA8 is over-expressed in multiple myeloma andcolonic carcinoma and BCR-ABL1-induced expression of HSPA8 promotes cellsurvival in chronic myeloid leukemia (Chatterjee et al., 2013; Dadkhahet al., 2013; Jose-Eneriz et al., 2008; Kubota et al., 2010; Wang etal., 2013a).

HSPA8P8 is a pseudogene (RefSeq, 2002).

HSPA9 encodes heat shock 70 kDa protein 9. This protein plays a role incell proliferation, stress response and maintenance of the mitochondria(RefSeq, 2002). HSPA9 regulates cellular processes ranging from viralinfection to neurodegeneration, which also includes carcinogenesis(Flachbartova and Kovacech, 2013). HSPA9 is up-regulated inhepatocellular carcinoma and colorectal cancer (Rozenberg et al., 2013;Chen et al., 2014a; Kuramitsu and Nakamura, 2005). HSPA9 plays a role inthe development of gastric cancer (Ando et al., 2014). HSPA9 is apotential therapeutic target for improved treatment of drug-resistantovarian cancer (Yang et al., 2013).

IGDCC4 encodes immunoglobulin superfamily, DCC subclass, member 4 and islocated on chromosome 15q22.31 (RefSeq, 2002). GDCC4 is expressed inhepatocellular carcinoma (Joy and Burns, 1988; Marquardt et al., 2011).GDCC4 plays a role in acute lymphoblastic leukemia (Taylor et al.,2007).

IGF2BP3 encodes insulin-like growth factor II mRNA binding protein 3, anoncofetal protein, which represses translation of insulin-like growthfactor II (RefSeq, 2002). Several studies have shown that IGF2BP3 actsin various important aspects of cell function, such as cellpolarization, migration, morphology, metabolism, proliferation anddifferentiation.

In vitro studies have shown that IGF2BP3 promotes tumor cellproliferation, adhesion, and invasion. Furthermore, IGF2BP3 has beenshown to be associated with aggressive and advanced cancers (Bell etal., 2013; Gong et al., 2014). IGF2BP3 over-expression has beendescribed in numerous tumor types and correlated with poor prognosis,advanced tumor stage and metastasis, as for example in neuroblastoma,colorectal carcinoma, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, prostate cancer, and renal cell carcinoma (Bell et al., 2013;Findeis-Hosey and Xu, 2012; Hu et al., 2014a; Szarvas et al., 2014; Jenget al., 2009; Chen et al., 2011c; Chen et al., 2013; Hoffmann et al.,2008; Lin et al., 2013b; Yuan et al., 2009).

IPO5 encodes importin 5, a member of the importin beta family. Importinsare essential in the translocation of proteins through the nuclear porecomplex (RefSeq, 2002).

IPO7 encodes importin 7. The importin alpha/beta complex and the GTPaseRan mediate nuclear import of proteins with a classical nuclearlocalization signal (RefSeq, 2002). IPO7 is frequently over-expressed incancers (Golomb et al., 2012). IPO7 is dysregulated in glioblastoma,Hodgkin lymphoma and breast cancer (Jung et al., 2013; Ju et al., 2013;Nagel et al., 2014; Xue et al., 2015). IPO7 is a microRNA target that isdown-regulated in prostate carcinoma (Szczyrba et al., 2013). Elevatedlevels of IPO7 mRNA in colorectal carcinoma are associated withincreased proliferation (Li et al., 2000).

IQGAP3 encodes a member of the IQ-motif-containing GAP family which actsat the interface between cellular signaling and the cytoskeleton. IQGAP3regulates the Rac1/Cdc42-promoted neurite outgrowth and interactsdirectly with calmodulin and the myosin light chain (Wang et al., 2007;Atcheson et al., 2011). IQGAP3 is over-expressed in lung cancer and isassociated with tumor cell growth, migration and invasion. Furthermore,it is up-regulated by chromosomal amplification in hepatocellularcarcinoma and the expression of IQGAP3 is increased in p53-mutatedcolorectal cancer patients with poor survival (Katkoori et al., 2012;Yang et al., 2014b; Skawran et al., 2008). IQGAP3 is modulating theEGFR/Ras/ERK signaling cascade and interacts with Rac/Cdc42 (Yang etal., 2014b; Kunimoto et al., 2009).

KDELR1 encodes KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum proteinretention receptor 1. KDELR1 is structurally and functionally similar tothe yeast ERD2 gene product (RefSeq, 2002). KDELR1 has a role intumorigenesis (Yi et al., 2009). Decreased KDELR1 levels are found inhepatoma cells (Hou et al., 2015). Down-regulation of KDELR1 is seen inacute myeloid leukemia (Caldarelli et al., 2013).

KPNA2 encodes karyopherin alpha 2. KPNA2 may be involved in the nucleartransport of proteins (RefSeq, 2002). KPNA2 expression is dysregulatedin epithelial ovarian cancer (Lin et al., 2015). KPNA2 is down-regulatedin large oral squamous cell carcinoma tumors in comparison to smalltumors (Diniz et al., 2015). KPNA2 contributes to the aberrantlocalization of key proteins and to poor prognosis of breast cancer(Alshareeda et al., 2015). The expression of KPNA2 is significantlyup-regulated in the upper tract urothelial carcinoma and in endometrialcancers (Ikenberg et al., 2014; Shi et al., 2015). KPNA2 promotes tumorgrowth in hepatocellular carcinoma (Hu et al., 2014b).

KRT19 encodes a member of the keratin family. Keratins are intermediatefilament proteins responsible for the structural integrity of epithelialcells and are subdivided into cytokeratins and hair keratins. KRT19 isspecifically expressed in the periderm, the transiently superficiallayer that envelopes the developing epidermis (RefSeq, 2002). KRT19expression in tumor cells is a prognostic marker for several tumorentities such as breast, lung, ovarian and hepatocellular cancer(Skondra et al., 2014; Gao et al., 2014a; Liu et al., 2013a; Lee et al.,2013). KRT19 has been shown to be an independent prognostic factor forpancreatic neuroendocrine tumors, especially the insulin-negativetumors. KRT19 positive tumors are associated with poor outcomeirrespective of the established pathologic parameters such as size,mitoses, lymphovascular invasion, and necrosis (Jain et al., 2010).

KRT8 (also called CK8) encodes a member of type II keratin family thatdimerizes with keratin 18 to form an intermediate filament insingle-layered epithelial cells. KRT8 plays a role in maintainingcellular structural integrity and also has a function in signaltransduction and cellular differentiation (RefSeq, 2002). KRT8 isup-regulated and secreted from different cancer cells including lung,prostate and breast cancer. High levels of KRT8 correlate with increasedmigration and invasion (Gonias et al., 2001; Kuchma et al., 2012;Fukunaga et al., 2002; Takei et al., 1995). The MEK/ERK pathwayregulates sphingosylphosphorycholine-induced KRT8 phosphorylation atSer431. This leads to keratin cytoskeleton re-organization andconsequently enhances the migration of tumor cells (Busch et al., 2012).The tumor suppressor SMAR down-regulates KRT8 expression and this leadsto a decreased migration and invasiveness of cells (Pavithra et al.,2009; Mukhopadhyay and Roth, 1996).

KRT8P44 encodes keratin 8 pseudogene 44, which is located on chromosome6q26 (RefSeq, 2002).

MACC1 encodes a key regulator of the hepatocyte growth factor (HGF)receptor pathway which is involved in cellular growth,epithelial-mesenchymal transition, angiogenesis, cell motility,invasiveness and metastasis (RefSeq, 2002). MACC1 is over-expressed inmany cancer entities including gastric, colorectal, lung and breastcancer and is associated with cancer progression, metastasis and poorsurvival of patients (Huang et al., 2013b; Ma et al., 2013; Stein, 2013;Wang et al., 2015b; Wang et al., 2015m; Ilm et al., 2015). MACC1promotes carcinogenesis through targeting beta-catenin and PI3K/AKTsignaling pathways, which leads to an increase of c-Met and beta-cateninand their downstream target genes including c-Myc, cyclin D1, caspase9,BAD and MMP9 (Zhen et al., 2014; Yao et al., 2015).

MAGED2 encodes melanoma antigen family D, 2, a member of a new definedMAGE-D cluster in Xp11.2, a hot spot for X-linked mental retardation.MAGED2 is expressed ubiquitously with high expression levels in specificbrain regions and in the interstitium of testes. MAGED2 is a potentialnegative regulator of wildtype p53 activity (Langnaese et al., 2001;Papageorgio et al., 2007). MAGED2 over-expression is associated withmelanoma, breast cancer and colon cancer (Li et al., 2004; Strekalova etal., 2015).

MAN2A1 encodes mannosidase alpha class 2A, member 1, which is localizedin the Golgi and catalyzes the final hydrolytic step in theasparagine-linked oligosaccharide maturation pathway (RefSeq, 2002).Swainsonine inhibits MAN2A1, resulting in the inhibition of theproduction of beta 1,6-branched N-linked glycans, which are related tothe malignant phenotype of tumor cells (Yagel et al., 1990;Gerber-Lemaire and Juillerat-Jeanneret, 2010; Santos et al., 2011;Przybylo et al., 2005; Dennis and Laferte, 1987; Baptista et al., 1994;Goss et al., 1994; Fujieda et al., 1994; Korczak and Dennis, 1993;Roberts et al., 1998; Goss et al., 1997; Goss et al., 1995; Seftor etal., 1991). A SNP in MAN2A1 is strongly associated with childhood acutelymphoblastic leukemia (Han et al., 2010).

MAP1A encodes microtubule associated protein 1A which is involved inmicrotubule assembly, an essential step in neurogenesis (RefSeq, 2002).MAP1A accumulates in retinoic acid-induced P19 embryonal carcinoma cells(Vaillant and Brown, 1995). MAP1A is down-regulated in thetumor-adjacent stroma of prostate cancer (Zhu et al., 2015b). MAP1A mayplay a role in cell proliferation (Matsuno et al., 2004). Danusertibsignificantly increases the expression level of membrane-bound MAP1A inbreast cancer (Li et al., 2015c). Baicalein up-regulates MAP1A in thehepatocellular carcinoma cell line HepG2 (Wang et al., 20151). MAP1A isinversely correlated to p62 in cutaneous squamous cell carcinoma(Yoshihara et al., 2014). Gamma-tocotrienol induces an increasedconversion of MAP1A from its cytosolic to its lipidated isoform (Tiwariet al., 2014).

MAT2A encodes methionine adenosyltransferase 2A which catalyzes theproduction of S-adenosylmethionine from methionine and ATP (RefSeq,2002). MAT2A is up-regulated in tamoxifen-resistant MCF-7 breast cancercells (Phuong et al., 2015). There are higher levels of sumoylated andtotal MAT2A in colon cancer. Interaction between Ubc9, Bcl2, and MAT2Aenhance growth and survival of cancer cells (Tomasi et al., 2015). MAT2Aexpression is down-regulated in renal cell carcinoma and in theS-adenosylmethionine-treated hepatocellular carcinoma cell line WCH17(Kuang et al., 2014; Wang et al., 2014b). The MAT1A:MAT2A switch isassociated with global DNA hypomethylation, decreased DNA repair,genomic instability, and signaling deregulation in hepatocellularcarcinoma (Woodburn et al., 2013; Frau et al., 2013). MAT2A isup-regulated in hepatocellular cell carcinoma, gastric cancer, and coloncancer (Frau et al., 2012; Zhang et al., 2013e; Tomasi et al., 2013;Frau et al., 2013; Lo et al., 2013). MAT2A is correlated with tumorclassification, lymph node metastasis, and poor tumor differentiation ingastric cancer patients (Liu et al., 2011b; Zhang et al., 2013e). MAT2Ais a transcriptional co-repressor of the oncoprotein MafK (Katoh et al.,2011). MAT2A is linked to tumor growth and progression in liver cancer(Vazquez-Chantada et al., 2010; Liu et al., 2011a; Lu and Mato, 2008).

MBTPS2 is a membrane-embedded zinc metalloprotease that activatessignaling of proteins involved in sterol control of transcription andplays a role in ER stress response (Oeffner et al., 2009).

MCM4 encodes the minichromosome maintenance complex component 4 which isessential for the initiation of eukaryotic genome replication (RefSeq,2002). MCM4 expression is associated with up-regulated carbonicanhydrase IX, a transmembrane glycoprotein which is correlated withdecreased survival and cancer progression in several entities includingesophageal cancer (Huber et al., 2015). Has-miR-615-3p may be involvedin nasopharyngeal carcinoma by regulating MCM4 (Chen et al., 2015b).MCM4 might play a role in the development of bladder cancer (Zekri etal., 2015). A gain-of-function mutant of p53 increases the expression ofMCM4 in breast cancer (Polotskaia et al., 2015). There is a mutation ofMCM4 in human skin cancer which shows reduced DNA helicase activity(Ishimi and Irie, 2015). MCM4 over-expression alone is only weaklyassociated with shorter survival in breast cancer. Over-expression ofall six parts of the MCM complex is strongly associated with shortersurvival (Kwok et al., 2015). MCM4 is differentially expressed in lungadenocarcinoma and laryngeal squamous cell carcinoma (Lian et al., 2013;Zhang et al., 2014c). MCM4 is significantly over-expressed in cervicalcancer (Das et al., 2013; Das et al., 2015). MCM4 may be used as abiomarker for colorectal cancer (Fijneman et al., 2012).

MIER1 (also called MI-ER1) encodes a transcriptional regulator that wasfirst identified in Xenopus leavis (RefSeq, 2002). MIER1 is up-regulatedin chronic myeloid leukemia (CML) and breast cancer, where loss of thenuclear transcript variant alpha is associated with cancer progressionand proliferation (McCarthy et al., 2008; Ding et al., 2003; Mascarenhaset al., 2014). The transcriptional repressor MIER1 functions due tointeraction with HDAC1 (Ding et al., 2003).

MIR2861 is a short non-coding RNAs that is involved inpost-transcriptional regulation of gene expression by affecting both thestability and translation of mRNAs (RefSeq, 2002). MIR2861 expression isup-regulated in papillary thyroid carcinoma (PTC) with lymph nodemetastasis in comparison to PTC without lymph node metastasis (Wang etal., 2013f).

MLEC encodes malectin, which is a type I membrane-anchored ER protein.MLEC has an affinity for Glc2Man9GlcNAc2 (G2M9) N-glycans and isinvolved in regulating glycosylation in the ER. MLEC has also been shownto interact with ribophorin I and may be involved in directing thedegradation of misfolded proteins (RefSeq, 2002; Pierce and Taniguchi,2009). MLEC is de-regulated in colorectal cancer and enhanced inglioblastoma (Sethi et al., 2015; Demeure et al., 2016). MLEC might be abiomarker for thyroid papillary carcinoma (Ban et al., 2012).

MVP encodes the major compartment of the vault complex, a protein whichmay play a role in multiple cellular processes by regulating MAPK,JAK/STAT and PI3K/Akt signaling pathways. It also plays a role inmultidrug resistance, innate immunity, cell survival anddifferentiation, and expression of this gene may be a prognostic markerfor several types of cancer (RefSeq, 2002; Tucci et al., 2009; Lara etal., 2011; Scagliotti et al., 1999; van den Heuvel-Eibrink M M et al.,2000; Perez-Tomas, 2006; Scheffer et al., 2000; Ramachandran, 2007;Sekine et al., 2007; Lu and Shervington, 2008). MVP is highly expressedin several central nervous system tumors (Yang et al., 2012a). MVP ishighly expressed in cancer, and in several chemoresistant cancer celllines (Szaflarski et al., 2011; Mossink et al., 2003). MVP expressionlevel increases with age and facilitates apoptosis resistance (Ryu andPark, 2009).

MYBBP1A (also called p160) encodes a nucleolar transcriptional regulatorthat was first identified by its ability to bind to the Mybproto-oncogene protein. MYBBP1A might play a role in many cellularprocesses, including response to nucleolar stress, tumor suppression andsynthesis of ribosomal DNA (RefSeq, 2002). MYBBP1A is de-regulated indifferent cancer entities, including lung, breast and head and neckcancer. It is associated with cell proliferation and metastasis(Bidkhori et al., 2013; George et al., 2015; Acuna Sanhueza et al.,2012; Akaogi et al., 2013). MYBBP1A promotes transcriptional activityvia p53 activation as well as Myb binding and regulates cell cycle andmitosis leading to G2/M arrest or anomalous mitosis by affecting thecontrol of chromosomal segregation (Tavner et al., 1998; Tsuchiya etal., 2011; Mori et al., 2012; Ono et al., 2013).

NCAPD2 (also called CNAP1) encodes non-SMC condensin I complex subunitD2 that is involved in chromosome condensation and associated withAlzheimer's disease (Ball, Jr. et al., 2002; Zhang et al., 2014b).NCAPD2 over-expression was found in the development of ovarian cancertogether with its amplification and mutation during tumor progression(Emmanuel et al., 2011).

NCAPG encodes the non-SMC condensing I complex subunit G which isresponsible for the condensation and stabilization of chromosomes duringmitosis and meiosis (RefSeq, 2002). NCAPG is down-regulated in patientswith multiple myeloma, acute myeloid leukemia, and leukemic cells fromblood or myeloma cells (Cohen et al., 2014). NCAPG may be a multi-drugresistant gene in colorectal cancer (Li et al., 2012). NCAPG is highlyup-regulated in the chromophobe subtype of human cell carcinoma but notin conventional human renal cell carcinoma (Kim et al., 2010a).Up-regulation of NCAPG is associated with melanoma progression (Ryu etal., 2007). NCAPG is associated with uveal melanoma (Van Ginkel et al.,1998). NCAPG shows variable expression in different tumor cells (Jageret al., 2000).

NLE1 encodes a notchless homolog and member of the WD40-repeat proteinfamily that is involved in embryonic development through differentsignal pathways and seems to play a role in ribosome maturation(Beck-Cormier et al., 2014; Romes et al., 2016; Lossie et al., 2012).

NOMO1 (also called PM5) encodes Nodal modulator 1, a protein that mightbe part of a protein complex that participates in the Nodal signalingpathway during vertebrate development (RefSeq, 2002). NOMO1 isde-regulated in prostate cancer and in T-cell lymphoma cells (Stubbs etal., 1999; Lange et al., 2009).

NOMO2 encodes Nodal modulator 2, a protein that might be part of aprotein complex that participates in the Nodal signaling pathway duringvertebrate development (RefSeq, 2002). NOMO2 is up-regulated at theepithelium/stroma cell interface in the transition to cervicalintraepithelial neoplasia (CIN) 3 and cervical cancer as part of apro-invasive genomic signature that may be a response to epithelialtumor cell over-crowding (Gius et al., 2007).

NOMO3 encodes Nodal modulator 3, a protein that might be part of aprotein complex that participates in the Nodal signaling pathway duringvertebrate development (RefSeq, 2002). NOMO3 is de-regulated by DNAmethylation in non-small cell lung cancer (Mullapudi et al., 2015).NOMO3 is an enriched membrane protein associated with glycosylation inovarian cancer tissues (Allam et al., 2015).

NONO (also known as p54nrb) encodes non-POU domain containing,octamer-binding. NONO is an RNA-binding protein which plays variousroles in the nucleus, including transcriptional regulation and RNAsplicing. A rearrangement between this gene and the transcription factorE3 has been observed in papillary renal cell carcinoma (RefSeq, 2002;Macher-Goeppinger et al., 2012). NONO expression strongly correlateswith vascular invasion and decreased survival (Barboro et al., 2008).Furospinosulin selectively inhibits the growth of hypoxia-adapted cancercells, maybe through direct binding to NONO (Arai et al., 2016). NONOmediates MIA/CD-RAP action to promote chondrogenesis and progression ofmalignant melanoma (Schmid et al., 2013). NONO expression correlateswith the expression of c-Myc, cyclin D1, and CDK4 (Nelson et al., 2012).Knock-out of NONO in YB-1 over-expressing colorectal cancers cansensitize them to oxaliplatin (Tsofack et al., 2011). Simvastatinstrongly down-regulates NONO and reduces melanoma progression (Schiffneret al., 2011; Zanfardino et al., 2013). NONO is over-expressed in breastcancer and melanoma (Schiffner et al., 2011; Zhu et al., 2015d). NPC1encodes Niemann-Pick disease, type C1, a large protein that resides inthe limiting membrane of endosomes and lysosomes and mediatesintracellular cholesterol trafficking via binding of cholesterol to itsN-terminal domain (RefSeq, 2002). The promotor of NPC1 ishypo-methylated and NPC1 expression is up-regulated in esophageal cancer(Singh et al., 2015). NPC1 is differentially expressed in isogenicmetastatic cancer cell lines, human embryonic stem cells, and humanembryonal carcinoma cells (Lund et al., 2015; Dormeyer et al., 2008).NPC1 degradation is regulated by Akt. Thus NPC1 is linked to cellproliferation and migration in cervical cancer (Du et al., 2015).Treatment with sildenafil reduces NPC1 expression and kills brain cancerstem cells (Booth et al., 2015). Inhibitors of cholesterol metabolism,including NPC1 for cholesterol uptake, are thought to be beneficial forcancer treatment (Ali-Rahmani et al., 2014). NPC1 is up-regulated inTNF-alpha-resistant MCF-7 breast adenocarcinoma cells (Vincent et al.,2010; Moussay et al., 2011).

NPC2 encodes a protein with a lipid recognition domain that may functionin regulating the transport of cholesterol through the lateendosomal/lysosomal system. Mutations in this gene are associated withNiemann-Pick disease and frontal lobe atrophy (RefSeq, 2002). NPC2 isde-regulated in different cancer entities, including breast, colon,lung, kidney and liver cancer (McDonald et al., 2004; Garcia-Lorenzo etal., 2012; Liao et al., 2013). NPC-related cholesterol perturbationinduces abnormal signaling pathways leading to p38 MAPK activation,Mdm2-mediated p53 degradation, ROCK activation and increased RhoAsynthesis (Qin et al., 2010).

NUP160 encodes a nucleoporin of 160 kDa that is part of the nuclear porecomplex that mediates the nucleoplasmic transport (RefSeq, 2002).NUP160-SLC43A3 is a recurrent fusion oncogene in angiosarcoma andassociated with tumor progression (Shimozono et al., 2015).

NUP205 encodes nucleoporin 205 kDa (RefSeq, 2002). NUP205 is stabilizedby TMEM209. This interaction is a critical driver for lung cancerproliferation (Fujitomo et al., 2012).

NUP98 encodes nucleoporin 98 kDa which participates in many cellularprocesses, including nuclear import, nuclear export, mitoticprogression, and regulation of gene expression. Translocations betweenthis gene and many other partner genes have been observed in differentleukemias. Rearrangements typically result in chimeras with theN-terminal GLGF domain of this gene to the C-terminus of the partnergene (RefSeq, 2002). NUP98 rearrangement induces leukemia in mice. Itenhances proliferation and disrupts differentiation in primary humanhematopoietic precursors (Takeda and Yaseen, 2014). Dys-regulation ofhomeobox genes, which cause NUP98 rearrangement, result in leukemictransformation (Gough et al., 2011; De et al., 2014; Slape and Aplan,2004; Grier et al., 2005; Abramovich et al., 2005; Nakamura, 2005;Shimada et al., 2000; Argiropoulos and Humphries, 2007). NUP98rearranges with several partners in hematopoietic malignancies,including acute myeloid leukemia, chronic myeloid leukemia in blastcrisis, myelodysplastic syndrome, acute lymphoblastic leukemia, andbilineage/biphenotypic leukemia (Tosic et al., 2009; Haznedaroglu andBeyazit, 2010; Shi et al., 2011; Gough et al., 2011; Panagopoulos etal., 2003; Morerio et al., 2006; Moore et al., 2007; Ahuja et al., 2001;McCormack et al., 2008; Lam and Aplan, 2001). NUP98 is linked totumorigenesis (Xu and Powers, 2009; Simon and Rout, 2014). NUP98 is amodulator of genomic stability and a suppressor of tumor development(Rao et al., 2009).

OXSR1 encodes a the Ser/Thr protein kinase that regulates down-streamkinases in response to oxidative stress and may play a role inregulating the actin cytoskeleton (RefSeq, 2002). OXSR1 is up-regulatedin the tumor stroma from human breast cancer patients and associatedwith recurrence (Pavlides et al., 2010).

PCSK9 encodes a member of the subtilisin-like proprotein convertasefamily, which includes proteases that process protein and peptideprecursors trafficking through regulated or constitutive branches of thesecretory pathway. It plays a role in cholesterol and fatty acidmetabolism (RefSeq, 2002). PCSK9 is de-regulated in different cancerentities including liver, lung and gastric cancer (Bhat et al., 2015;Marimuthu et al., 2013; Demidyuk et al., 2013). PCSK9 deficiency reducesliver metastasis by its ability to lower cholesterol levels and byenhancing TNFalpha-mediated apoptosis. Other studies show in contrast noeffect of cholesterol levels on cancer risk (Folsom et al., 2007; Sun etal., 2012).

PDAP1 encodes a phosphoprotein that may up-regulate the PDGFA-stimulatedgrowth of fibroblasts and also down-regulate the mitogenicity of PDGFB(RefSeq, 2002). PDAP1 is over-expressed in different cancer types,including gastric and rectal cancer, and could thereby play a role as abiomarker (Choi et al., 2011; Marimuthu et al., 2013).

PDIA3 (also known as ERp57) encodes the protein disulfide isomerasefamily A member 3, a protein of the endoplasmic reticulum that interactswith lectin chaperons, calreticulin, and calnexin to modulate folding ofnewly synthesized glycoproteins (RefSeq, 2002; Coe and Michalak, 2010).PDIA3 may be used as a biomarker and in the diagnosis of tumors(Shishkin et al., 2013). PDIA3 is differentially expressed in gliomas(Deighton et al., 2010). PDIA3 is implicated in human pathologyincluding cancer and Alzheimer's disease (Coe and Michalak, 2010). PDIA3is an auxiliary factor of TAP which loads viral and self-peptides on MHCclass I (Coe and Michalak, 2010; Abele and Tampe, 2011).

PFDN1 encodes prefoldin subunit 1, one of six subunits of prefoldin, amolecular chaperone complex that binds and stabilizes newly synthesizedpolypeptides, thereby allowing them to fold correctly (RefSeq, 2002).PFDN1 is involved in colorectal cancer progression, and is positivelycorrelated with tumor size and invasion (Wang et al., 2015e). PFDN1 isup-regulated in several cancers including colorectal cancer (Wang etal., 2015e). PFDN1 can be used as a reference gene in nasopharyngealcarcinoma (Guo et al., 2010).

PHB encodes prohibitin which is proposed to play a role in humancellular senescence and tumor suppression (RefSeq, 2002; Mishra et al.,2010; Theiss and Sitaraman, 2011; Zhou and Qin, 2013; Mishra et al.,2005; McClung et al., 1995; Rajalingam and Rudel, 2005). PHB activatesthe Raf/MEK/ERK pathway which is involved in cell growth and malignanttransformation (Rajalingam and Rudel, 2005). PHB is a potentialbiomarker in nasopharyngeal carcinoma that predicts the treatmentresponse to radiotherapy (Chen et al., 2015e). PHB was identified in theproteomic analysis of drug-resistant cancer cells, drug action, anddisease state tissues (Guo et al., 2013). PHB is over-expressed in manycancer entities (Zhou and Qin, 2013). The core protein of hepatitis Cvirus, which is a major risk factor for hepatocellular carcinoma,induces over-production of oxidative stress by impairing prohibitin(Theiss and Sitaraman, 2011; Schrier and Falk, 2011; Koike, 2014). PHBis differentially expressed in gliomas (Deighton et al., 2010).

PKM2 encodes pyruvate kinase, muscle, a protein involved in glycolysis.PKM2 interacts with thyroid hormone and thus may mediate cellularmetabolic effects induced by thyroid hormones. It is also thought to beinvolved in bacterial pathogenesis (RefSeq, 2002; Israelsen and VanderHeiden, 2015). PKM2 was shown to be crucial for cancer cellproliferation and tumor growth (Chen et al., 2014b; Li et al., 2014c;DeLaBarre et al., 2014). N-myc acts as a transcriptional regulator forPKM2 in medulloblastoma (Tech et al., 2015). PKM2 seems to play a rolein hepatocarcinogenesis, epithelial mesenchymal transition, andangiogenesis (Nakao et al., 2014). PKM2 is one of the two key factors ofthe Warburg effect in oncology (Tamada et al., 2012; Warner et al.,2014; Ng et al., 2015). Expression of PKM2 is up-regulated in cancercells (Chaneton and Gottlieb, 2012; Luo and Semenza, 2012; Wu and Le,2013). In malignant cells PKM2 functions in glycolysis, as atranscriptional coactivator and as a protein kinase. In the latterfunction it translocates to the nucleus and phosphorylates histone 3which finally causes the progress of the cell cycle in glioblastomas(Semenza, 2011; Luo and Semenza, 2012; Tamada et al., 2012; Venneti andThompson, 2013; Yang and Lu, 2013; Gupta et al., 2014; Iqbal et al.,2014; Chen et al., 2014b; Warner et al., 2014). The low-activity-dimericPKM2 might play a role in cancer instead of the active tetrameric form(Mazurek, 2011; Wong et al., 2015; Iqbal et al., 2014; Mazurek, 2007).

PKP3 encodes plakophilin, 3 a member of the arm-repeat and plakophilinfamily, which is localized to desmosomes and nuclei and participates inlinking cadherins to intermediate filaments in the cytoskeleton. PKP3may act in cellular desmosome-dependent adhesion and signaling pathways(RefSeq, 2002). Increased PKP3 mRNA in the blood of gastrointestinalcancer patients can be used as a biomarker and predictor for diseaseoutcome (Valladares-Ayerbes et al., 2010). Over-expression of PKP3 wascorrelated with a poor outcome in breast, lung and prostate cancer,whereas down-regulation in bladder cancer is linked to invasive behavior(Furukawa et al., 2005; Breuninger et al., 2010; Demirag et al., 2012;Takahashi et al., 2012). Loss of PKP3 leads to increased protein levelsof MMP7 and PRL3, which are required for cell migration and tumorformation (Khapare et al., 2012; Basu et al., 2015b).

PLEC encodes the plakin family member plectin, a protein involved in thecross-linking and organization of the cytoskeleton and adhesioncomplexes (Bouameur et al., 2014). PLEC is over-expressed in colorectaladenocarcinoma, head and neck squamous cell carcinoma and pancreaticcancer (Lee et al., 2004; Katada et al., 2012; Bausch et al., 2011).

PLXNA2 encodes plexin A2 which is a semaphorin co-receptor. PLXNA2 isthought to transduce signals from semaphorin 3A and 3C (RefSeq, 2002).KIAA1199 binds to PLXNA2, resulting in the inhibition of semaphorin 3Amediated cell death via EGFR stabilization and signaling (Shostak etal., 2014). PLXNA2 is up-regulated in TMPRSS2-ERG-positive prostatecancer and metastatic prostate cancer, resulting in enhanced cellmigration and invasion (Tian et al., 2014). PLXNA2 has higher expressionlevels in more aggressive breast cancer and is associated withtumorigenesis (Gabrovska et al., 2011).

POLA2 encodes an accessory subunit of DNA polymerase alpha (also called70/68 kDa or B subunit) that plays an important role in the initiationof DNA replication by tethering the catalytic subunit A and the primasecomplex (Collins et al., 1993; Pollok et al., 2003). POLA2 isde-regulated in different cancer types including gastrointestinalstromal tumors and non-small cell lung cancer (Mah et al., 2014; Kang etal., 2015). During S-phase, POLA2 is attached to telomeres. It isassociated with telomerase activity and is important for propertelomeric overhang processing through fill-in synthesis (Diotti et al.,2015). PPM1G encodes protein phosphatase, Mg2+/Mn2+ dependent, 1G. Thisprotein is found to be responsible for the dephosphorylation of pre-mRNAsplicing factors, which is important for the formation of functionalspliceosomes (RefSeq, 2002). PPM1 G regulates the E3 ligase WVVP2 whichdifferentially regulates cellular p73 and DeltaNp73 (Chaudhary andMaddika, 2014). PPM1G is able to bind apoptosis-stimulating proteins ofp53 which are uniquely over-expressed in various entities (Skene-Arnoldet al., 2013). PPM1G down-regulates USP7S by dephosphorylation,resulting in p53 accumulation (Khoronenkova et al., 2012).

PPP1R15B encodes a protein phosphatase-1 (PP1) interacting protein.PPP1R15B promotes de-phosphorylation of the transcription initiationfactor EIF2-alpha through recruitment of PP1 catalytic subunits (RefSeq,2002). Down-regulation of PPP1R15B results in impaired proliferation dueto unsuccessful transition from G1 to S phase of the cell cycle,induction of apoptosis by increased activity of caspase 3/7, andregulation of ERalpha activity (Shahmoradgoli et al., 2013).

PPY encodes a protein that is synthesized as a 95 amino acid polypeptideprecursor in the pancreatic islets of Langerhans. It is cleaved into twopeptide products; the active hormone of 36 amino acids and anicosapeptide of unknown function. The hormone acts as a regulator ofpancreatic and gastrointestinal functions and may be important in theregulation of food intake (RefSeq, 2002). Patients with diabetes melitussecondary to pancreatic cancer have a blunted PPY response to a mixedmeal compared to patients with type 2 diabetes melitus. However, theblunted PPY response is only observed in those pancreas carcinomapatients with a tumor located in the head of the pancreas (Hart et al.,2015).

PRKDC encodes the catalytic subunit of the DNA-dependent protein kinase(DNA-PK) (RefSeq, 2002). PRKDC is a frequently mutated gene inendometriosis-associated ovarian cancer and breast cancer (Er et al.,2016; Wheler et al., 2015). PRKDC is up-regulated in cancerous tissuescompared with normal tissues in colorectal carcinoma. Patients with highPRKDC expression show poorer overall survival (Sun et al., 2016b). PSEN1encodes presenilin 1 which is linked to Alzheimer's disease. It is partof the gamma-secretase complex which is required for Notch activation(RefSeq, 2002; Ponnurangam et al., 2015). Over-expression of PSEN1 by asmall interfering RNA sensitizes chemoresistant bladder cancer cells todrug-triggered cell death (Deng et al., 2015a). PSEN1 plays a key rolein epithelial-mesenchymal transition and chemoresistance bydown-regulating E-cadherin (Sehrawat et al., 2014; Dinicola et al.,2016). TRAF6-mediated PSEN1 activation results in promotion of tumorinvasiveness (Gudey et al., 2014; Sundar et al., 2015). Down-regulatedexpression of the gamma-secretase complex is thought to be a risk factorfor breast cancer specific mortality (Peltonen et al., 2013). PSEN1 isdifferentially expressed in T-cell acute lymphoblastic leukemia causedby dys-regulated Notch1 (Paryan et al., 2013). PSEN1 is over-expressedin oral squamous cell carcinoma cell lines and primary oralkeratinocytes isolated from oral squamous cell carcinoma tissue. PSEN1over-expression results in reduced cell adhesion in oral squamous cellcarcinoma by affecting P-cadherin (Bauer et al., 2013). Theendocannabinoid anandamide increases the expression and recruitment ofPSEN1 in cholangiocarcinoma (Frampton et al., 2010). p53 is able toregulate PSEN1 expression (Checler et al., 2010). PSEN1 is involved intumor reversion (Telerman and Amson, 2009).

PSEN2 encodes presenilin 2 which is linked to Alzheimer's disease. It ispart of the gamma-secretase complex which is required for Notchactivation (RefSeq, 2002). Oxidative stress and p53 expression level isincreased in PC12 cells carrying a mutated PSEN2 gene (Nguyen et al.,2007). PSEN2 is a useful prognostic factor in breast cancer. The novelPSEN2 alleles R62H and R71W affect PSEN2 function and may potentiallyconfer a moderate risk of susceptibility to breast cancer (To et al.,2006; Xu et al., 2006). PSEN2 is part of a 10-gene signature set whichis associated with recurrence-free survival time but not overallsurvival time in ovarian carcinoma (Chen and Liu, 2015). Loss of PSEN2may cause lung tumor development by up-regulating iPLA2 (Yun et al.,2014). Down-regulated expression of the gamma-secretase complex isthought to be a risk factor for breast cancer specific mortality(Peltonen et al., 2013). PSEN2 is differentially expressed inmegakaryocytic leukemia and gastric cancer. PSEN2 expression correlateswith tumor type, UICC tumor stage, tumor grade, and patient survival(Warneke et al., 2013; Hao et al., 2006). The promotor of PSEN2 isde-methylated in glioma tissues, causing PSEN2 over-expression (Liu etal., 2012). 2-arachidonylglycerol increases the expression andrecruitment of PSEN2 in cholangiocarcinoma (Frampton et al., 2010).PSEN2 causes tumor cell proliferation in rat pancreatic cancer bycleaving EpC (Maetzel et al., 2009; Thuma and Zoller, 2013).

PTGS1 (also known as Cox1) encodes the prostaglandin-endoperoxidesynthase 1 (prostaglandin G/H synthase and cyclooxygenase). PTGS1 isconstitutively expressed and catalyzes the conversion of arachinodate toprostaglandin. The encoded protein regulates angiogenesis in endothelialcells, and is inhibited by non-steroidal anti-inflammatory drugs, suchas aspirin. Based on its ability to function as both a cyclooxygenaseand as a peroxidase, PTGS1 has been identified as a moonlightingprotein. The protein may promote cell proliferation during tumorprogression (RefSeq, 2002; Tietz et al., 2013). PTGS1 may be involved intumorigenesis (Rouzer and Marnett, 2009). Enhanced tumor growth issupported by up-regulation of PTGS1 which plays a role in prostaglandinand VEGF production (Campione et al., 2015). PTGS1 is associated withdecreased survival for recurrent minor salivary gland carcinoma(Haymerle et al., 2015). PTGS1 is associated with breast carcinogenesis(Basu et al., 2015a; Serra et al., 2016). PTGS1 is frequentlyde-regulated in the progression of cancer (Karnezis et al., 2012).Deletion of PTGS1 results in robust decrease of basal cell carcinoma(Arbiser, 2010). Aspirin inhibits PTGS1-induced platelet activationwhich is thought to be involved in the development of inflammation andcancer, including colorectal carcinoma, head and neck cancer,gastrointestinal cancer, and pancreatic cancer (Pereira et al., 2009;Perrone et al., 2010; Schror, 2011; Garcia Rodriguez et al., 2013; Brunoet al., 2012; Yue et al., 2014; Sostres et al., 2014; Schror and Rauch,2013; Guillem-Llobat et al., 2014; Patrignani and Patrono, 2015;Patrono, 2015; Dovizio et al., 2015; Jimenez et al., 2007; Klass andShin, 2007).

PTGS2 (also called COX-2) encodes prostaglandin-endoperoxide synthase 2(cyclooxygenase), the key enzyme in prostaglandin biosynthesis that actsas a dioxygenase and as a peroxidase (RefSeq, 2002). Expression of PTGS2and prostaglandins is associated with various cancer types includingbreast, lung, gastric, pancreatic, colorectal and prostate tumors. Theexpression level is also directly proportional to tumor aggressivenessincluding metastasis (Shao et al., 2012; Kunzmann et al., 2013; Misraand Sharma, 2014; Aziz and Qiu, 2014; Thill et al., 2014; Knab et al.,2014; Huang and Huang, 2014; Wang et al., 2014c). Anti-inflammatoryagents with activity against PTGS2 have a strong potential for thechemoprevention of cancer (Harris, 2009; Ghosh et al., 2010).

PTPN14 encodes protein tyrosine phosphatase, non-receptor type 14, whichappears to regulate lymphatic development in mammals. A loss-of-functionmutation has been found in kindred with a lymphedema-choanal atresia(RefSeq, 2002). PTPN14 induces TGF-beta signaling, regulatesendothelial-mesenchymal transition, and organogenesis (Wyatt andKhew-Goodall, 2008). PTPN14 is down-regulated in cholangiocarcinoma andis inversely correlated with clinical-pathological features and survival(Wang et al., 2015d; Wang et al., 2015c). PTPN14 inhibits trafficking ofsoluble and membrane-bound proteins, resulting in prevention ofmetastasis (Belle et al., 2015). PTPN14 negatively regulates theoncoprotein Yes-associated protein (YAP), a key protein in the Hippopathway, which is responsible for organ size and tumorigenesis (Liu etal., 2013b; Huang et al., 2013a; Lin et al., 2013a). Loss-of-functionmutations in PTPN14 are involved in neuroblastoma relapse, breastcancer, and colorectal cancer (Laczmanska and Sasiadek, 2011; Wang etal., 2004; Schramm et al., 2015; Wyatt and Khew-Goodall, 2008).

RABGGTB is the beta subunit of Rab geranylgeranyltransferase thatcatalyzes the posttranslational geranylgeranylation of Rab GTPases(Pylypenko et al., 2003). RABGGTB is over-expressed inchemotherapy-refractory diffuse large B-cell lymphoma (Linderoth et al.,2008).

RAC1 encodes the ras-related C3 botulinum toxin substrate 1 (rho family,small GTP binding protein Rac1), a GTPase which belongs to the RASsuperfamily of small GTP-binding proteins. Members of this superfamilyappear to regulate a diverse array of cellular events, including thecontrol of cell growth, cytoskeletal reorganization, and the activationof protein kinases (RefSeq, 2002). RAC1 is important for neural crestdevelopment and can prevent melanoma formation (Shakhova, 2014). RAC1can be activated by the hepatocyte growth factor and the Met tyrosinekinase receptor, resulting in proliferation and migration of endothelialcells (Barrow-McGee and Kermorgant, 2014; Gallo et al., 2015). RAC1induces ROS in the viral oncogenesis of Kaposi's sarcoma (Mesri et al.,2013). RAC1 is involved in melanoma initiation and progression, inbreast cancer, and in head and neck cancer (Alan and Lundquist, 2013;Imianitov, 2013; Meierjohann, 2014). Tiam1 is able to regulate RAC1,which in turn regulates signaling pathways involved in cytoskeletalactivity, cell polarity, endocytosis and membrane trafficking, cellmigration, adhesion and invasion, cell growth and survival, metastasis,angiogenesis, and carcinogenesis (Bid et al., 2013; Boissier andHuynh-Do, 2014). RAC1 is thought to be an oncogene (Kunz, 2013; Kunz,2014). Mutations in RAC1 can cause a variety of disorders, includingmalignant transformation (Read, 2013; Chi et al., 2013). Activation ofRac1 results in formation of actin stress fibers, membrane ruffles,lamellipodia, and filopodia (Klopocka et al., 2013; van and van Buul,2012; Lane et al., 2014). RAC1 is down-regulated in astrocytic tumors,but is over-expressed in medulloblastoma tumors (Khalil and EI-Sibai,2012).

RAS3 encodes ras-related C3 botulinum toxin substrate 3 (rho family,small GTP binding protein Rac3), a GTPase which belongs to the RASsuperfamily of small GTP-binding proteins. Members of this superfamilyappear to regulate a diverse array of cellular events, including thecontrol of cell growth, cytoskeletal reorganization, and the activationof protein kinases (RefSeq, 2002). Over-expression of RAC3 is associatedwith poor prognosis in endometrial carcinoma (Balmer et al., 2006). RAC3is a target of ARHGAP6 which acts as a tumor suppressor in cervicalcancer (Li et al., 2015b). RAC3 is involved in the organization of thecytoskeleton, cell migration, and invasion (Liu et al., 2015c). RAC3 isdifferentially expressed in leukemia and non-small cell lung cancer, andis involved in tumor growth (Tan and Chen, 2014; Liu et al., 2015c;Koldehoff et al., 2008). RAC3 is involved in the TGF-beta-induceddown-regulation of E-cadherin in esophageal cancer (Dong et al., 2014;Xu et al., 2007). Rac3 induces the Rac3/ERK-2/NF-kappaB signalingpathway that triggers breast cancer cell aggressiveness. Endogenous Racactivity correlates with high metastatic potential in breast cancercells (Gest et al., 2013; Baugher et al., 2005). RAC3 is up-regulated inseveral cancers, including leukemia, prostate cancer, and breast cancer(Fernandez Larrosa et al., 2012; Liu et al., 2015c; Culig and Bartsch,2006; Calaf and Roy, 2007; Engers et al., 2007; Colo et al., 2007a; Coloet al., 2007b). RAC3 is an NF-kappaB coactivator which regulates cyclinD1 expression (Rubio et al., 2012; Colo et al., 2007b). Over-expressionof RAC3 in ERalpha-positive breast cancer results in enhanced cellmigration (Walker et al., 2011; Rubio et al., 2006).

RAD54 encodes a protein belonging to the DEAD-like helicase superfamily.It shares similarity with Saccharomyces cerevisiae RAD54 and RDH54, bothof which are involved in homologous recombination and repair of DNA.This protein binds to double-stranded DNA, and displays ATPase activityin the presence of DNA. This gene is highly expressed in testis andspleen, which suggests active roles in meiotic and mitotic recombination(RefSeq, 2002). Homozygous mutations of RAD54B were observed in primarylymphoma and colon cancer (Hiramoto et al., 1999). RAD54B counteractsgenome-destabilizing effects of direct binding of RAD51 to dsDNA inhuman tumor cells (Mason et al., 2015).

RAI14 (also called NORPEG) encodes retinoic acid induced 14. The gene isdetected in retinal pigment epithelial cells where it is inducible byall-trans-retinoic acid that is ubiquitously expressed in human tissuesand may have a role in human testis development and spermatogenesis(Kutty et al., 2001; Yuan et al., 2005). RAI14 is de-regulated ingastric cancer and connected with cell proliferation. It is a prognosticmarker for relapse-free survival for lung and breast cancer patients(Zhou et al., 2015a; Hsu et al., 2013).

RBM19 encodes a nucleolar protein that contains six RNA-binding motifsand may be involved in ribosome biogenesis (RefSeq, 2002). RBM19 iswidely expressed in human colorectal carcinoma (Lorenzen et al., 2005).Mutational inactivation of RBM19 results in elevated p53 activity andincreased apoptosis in mice (Zhang et al., 2008; Deisenroth and Zhang,2010).

RPF1 (also called BXDC5) encodes a nucleolar RNA binding protein thatcontains a sigma(70)-like motif and is required for ribosome biogenesis(Wehner and Baserga, 2002).

RPL13A encodes a member of the L13P family of ribosomal proteins that isa component of the 60S ribosomal subunit. The encoded protein also playsa role in the repression of inflammatory genes as a component of theIFN-gamma-activated inhibitor of translation (GAIT) complex (RefSeq,2002). RPL13A is de-regulated in different cancer types includingprostate, liver and colorectal cancer (Kasai et al., 2003; Ohl et al.,2005; Yoon et al., 2006). Depletion of RPL13A causes significantreduction of methylation of ribosomal RNA and of cap-independenttranslation mediated by IRES elements derived from p27, p53 and SNAT2mRNAs (Chaudhuri et al., 2007).

RPL13AP20 encodes ribosomal protein L13a pseudogene that is located onchromosome 12p13.1 (Balasubramanian et al., 2009).

RPL13AP5 encodes a ribosomal protein L13a pseudogene that is located onchromosome 10q24.1 (Balasubramanian et al., 2009).

RPL34 encodes the ribosomal protein L34 which is a component of the 60Ssubunit. Over-expression of this gene has been observed in some cancercells (RefSeq, 2002). Over-expression of RPL34 results in the promotionof malignant proliferation in non-small cell lung cancer (Yang et al.,2016). RPL34 plays a critical role in cell proliferation, cell cycledistribution and apoptosis of human malignant gastric cells (Liu et al.,2015a). RPTOR (also known as RAPTOR) encodes the regulatory associatedprotein of mTOR, complex 1. The protein is a compartment of a signalingpathway that regulates cell growth in response to nutrient and insulinlevels. The protein positively regulates the down-stream effectorribosomal protein S6 kinase, and negatively regulates the mTOR kinase(RefSeq, 2002). In the absence of either tuberous sclerosis complex 1 or2, mTOR-RPTOR signaling gets constitutively activated, resulting inenhanced and de-regulated protein synthesis and cell growth (Avruch etal., 2005; Kwiatkowski and Manning, 2005). mTOR positively regulatescell growth and survival primarily through direct interaction with RPTOR(Sun, 2013). In complex with mTOR, RPTOR controls cap-dependenttranslation, and this function is essential for PI3K-initiatedoncogenesis (Vogt et al., 2010). Rapalogs are agents that primarilyinhibit the mTOR-RPTOR complex 1 (mTORC1, rapamycin-sensitive) and areused in breast cancer therapy (Wysocki, 2009; De et al., 2013; Vinayakand Carlson, 2013; Le et al., 2008).

SEC24D encodes SEC24 homolog D, COPII coat complex component. SEC24D hassimilarity to yeast Sec24p component of COPII. COPII is the coat proteincomplex responsible for vesicle budding from the ER. This gene productis implicated in the shaping of the vesicle, and also in cargo selectionand concentration. Mutations in this gene have been associated withCole-Carpenter syndrome, a disorder affecting bone formation, resultingin craniofacial malformations and bones that break easily (RefSeq,2002). The induction ratio of SEC24D is enhanced in the human prostatecancer cell line LNCaP (DePrimo et al., 2002; Zhao et al., 2004). SEC24Dcan be phosphorylated by Akt (Sharpe et al., 2011).

SEPT10 encodes a member of the septin family of filament-formingcytoskeletal GTPases. It is localized to the cytoplasm and nucleus anddisplays GTP-binding and GTPase activity (RefSeq, 2002). SEPT10 isdown-regulated in different cancer types including bladder, breast,liver, lung, pancreas and prostate cancer as well as melanoma andleukemia. It is associated with poor prognosis for survival (Kienle etal., 2010; Liu et al., 2010b).

SEPT11 encodes a member of the conserved septin family offilament-forming cytoskeletal GTPases that are involved in a variety ofcellular functions including cytokinesis and vesicle trafficking(RefSeq, 2002). SEPT11 is over-expressed in different cancer entitiesincluding brain, cervix, pancreas and prostate cancer, melanoma andleukemia (Liu et al., 2010b). Loss of heterozygosity (LOH) of SEPT11 isassociated with poor prognosis in hepatocellular carcinomas. A fusiontranscript with MLL has been identified in myeloid neoplasia (Huang etal., 2010; Cerveira et al., 2011).

SEPT8 encodes a member of the septin family of nucleotide bindingproteins which is highly conserved and plays a role in the regulation ofcytoskeletal organization and cytokinesis (RefSeq, 2002). SEPT8 isup-regulated in different cancer types including bladder, liver,pancreas and lung cancer as well as leukemia (Liu et al., 2010b).

SERPINB2 (also known as PAI2) encodes serpin peptidase inhibitor, cladeB (ovalbumin), member 2 and is located on chromosome 18q21.3. It is anon-conventional serine protease inhibitor (SERPIN) which influencesgene expression, cell proliferation and differentiation, and apoptosis(RefSeq, 2002; Medcalf and Stasinopoulos, 2005). SERPINB2 encodes serpinpeptidase inhibitor, clade B (ovalbumin), member 2, an inhibitor ofextracellular protease urokinase plasminogen activator and tissueplasminogen activator (Schroder et al., 2014). SERPINB2 is expressed ina number of different tumors. SERPINB2 expression is associated withfavorable prognosis in breast and pancreatic cancers, but poor prognosisin endometrial, ovarian, and colorectal cancers (Schroder et al., 2014).SERPINB2 is an invasion- and metastasis-related gene (Pucci et al.,2016). SERPINB2 regulates urokinase-type plasminogen activator (uPA)which triggers the conversion of plasminogen to plasmin. Plasmin is ableto degrade the extracellular matrix (ECM), an important process of tumorprogression (Gershtein and Kushlinskii, 1999; Ulisse et al., 2009;Berger, 2002; Baldini et al., 2012; Mekkawy et al., 2014; Andreasen etal., 2000). Degradation of the ECM results in tumor progression, tumormass expansion, tumor growth factor release, cytokine activation, tumorcell proliferation, migration, and invasion (Hildenbrand et al., 2010;Magdolen et al., 2003; Halamkova et al., 2012; Duffy, 2004; Mekkawy etal., 2014; Dass et al., 2008). Many tumors show a correlation betweenuPA system components and tumor aggressiveness and survival (Mekkawy etal., 2014; Duffy and Duggan, 2004; Han et al., 2005). High levels ofSERPINB2 decrease tumor growth and metastasis (Croucher et al., 2008).

SH3BP4 encodes the SH3-domain binding protein 4 which is involved incargo-specific control of clathrin-mediated endocytosis, specificallycontrolling the internalization of a specific protein receptor (RefSeq,2002). SH3BP4 expression is 7-fold increased in the retinoblastoma cellline Y79 (Khanobdee et al., 2004). Fibroblast growth factor receptor 10stimulation in SH3BP4-depleted cells causes a decreased cell migrationin breast cancer cells and the inhibition of epithelial branching inmouse lung explants (Francavilla et al., 2013).

SHCBP1 encodes a protein that associates with human centralspindlin andis one of the crucial factors involved in midbody organization andcytokinesis completion (Asano et al., 2014). SHCBP1 is up-regulated inhuman hepatocellular carcinoma. Targeting SHCBP1 inhibits cellproliferation in human hepatocellular carcinoma cell lines (Tao et al.,2013). Among 16 genes with concomitant genomic alterations, SHCBP1 maybe involved in tumorigenesis and in the processes of invasion andprogression from pre-invasive ductal carcinoma in situ to invasiveductal carcinoma (Colak et al., 2013).

SIGMAR1 (also called OPRS1 or SIG-1R) encodes a sigma non-opioidintracellular receptor that interacts with a variety of psychotomimeticdrugs, including cocaine and amphetamines. Mutations in this gene areassociated with a juvenile amyotrophic lateral sclerosis (RefSeq, 2002).SIGMAR1 is over-expressed in tumor cell lines and tumors of variouscancer tissues, including lung, colon, skin, and breast cancer. SIGMAR1over-expression is associated with cell proliferation (Vilner et al.,1995; Aydar et al., 2004; Aydar et al., 2006; Bem et al., 1991; Skrzyckiand Czeczot, 2013). SIGMAR1 promotes hERG/bet1-integrin signaling,triggers the activation of the PI3K/Akt pathway, and induces thephosphorylation of translational regulator proteins like p70S6K, S6 and4E-BP1. SIGMAR1 increases motility and VEGF secretion, thus enhancingthe aggressiveness of tumor cells (Crottes et al., 2016; Kim et al.,2012a). SLC16A3 encodes solute carrier family 16 member 3, aproton-linked monocarboxylate transporter (RefSeq, 2002). Most solidtumors are known to rely on glycolysis for energy production. High ratesof glycolysis result in an increased production of lactate which hasbeen associated with poor clinical outcome and direct contribution totumor growth and progression. SLC16A3 is one of few monocarboxylatetransporters which facilitate the lactate export in cancer cells (Dhupet al., 2012; Draoui and Feron, 2011). The SLC16A3 expression has beenassociated with poor prognosis in hepatocellular cancer patients andincreased cell proliferation, migration and invasion in cell lineexperiments (Gao et al., 2015). The functional involvement of SLC16A3 inthe tumorigenesis was shown in a subset of pancreatic cancer (Baek etal., 2014).

SLC1A4 (also known as ASCT1) encodes solute carrier family(glutamate/neutral amino acid transporter), member 4 which is located onchromosome 2p15-p13 (RefSeq, 2002). The hepatocellular carcinoma cellline C3A enhances SLC1A4 expression after cysteine deprivation (Lee etal., 2008b). SLC1A4 acts as a recruiter of amino acids in esophagealadenocarcinoma (Younes et al., 2000). Knock-down of ASCT2 enhancesSLC1A4 mRNA levels in human hepatoma cells (Fuchs et al., 2004).Activation of the v-myc myelocytomatosis viral oncogene homologue geneleads to an up-regulation of SLC1A4 in the human glioma cell line Hs683(Jiang et al., 2012). Glutamine deprivation does not lead to anup-regulation of SLC1A4 in neuroblastoma (Wasa et al., 2002).

SLC1A5 (also known as ASCT2) encodes solute carrier family(glutamate/neutral amino acid transporter), member 5, which is asodium-dependent neutral amino acid transporter that can act as areceptor for RD114/type D retrovirus (RefSeq, 2002). c-Myc activationincreases SLC1A5 expression (Perez-Escuredo et al., 2016).Over-expression of SLC1A5 is associated with poor prognosis inclear-cell renal cell carcinoma (Liu et al., 2015d). A high expressionof CD147 is significantly associated with SLC1A5 in patients withpancreatic cancer (Kaira et al., 2015). SLC1A5 might be a biomarker fornon-small cell lung cancer (Hassanein et al., 2015; Hassanein et al.,2016). The ubiquitin ligase RNF5 regulates SLC1A5 in breast cancer (Jeonet al., 2015). SLC1A5 is over-expressed in several cancer entities,including advanced laryngeal cancer, prostate cancer, and adenoid cysticcarcinoma (Koo and Yoon, 2015; Wang et al., 2015f; Bhutia et al., 2015;Nikkuni et al., 2015; Ganapathy et al., 2009). Inhibition of SLC1A5 inbreast cancer leads to reduced glutamine uptake and proliferation (Chenet al., 2015d; van et al., 2015). SLC1A5 may stimulate tumor growth byregulating mTOR (Nakanishi and Tamai, 2011; Fuchs and Bode, 2005; Corbetet al., 2016; McGivan and Bungard, 2007).

SLC26A6 encodes a member of the solute carrier family 26 which consistsof anion transport proteins. SLC26A6 is involved in transportingchloride, oxalate, sulfate and bicarbonate ions (RefSeq, 2002).Mutations of SLC26A6 have been identified in different colorectal cancercell lines (Donnard et al., 2014). SLC26A6 gene expression and promoteractivity are inhibited by IFN-gamma (Saksena et al., 2010).

SLC52A3 (also called RFT2 or C20orf54) encodes a member of the solutecarrier family 52. It is a riboflavin transporter protein that likelyplays a role in intestinal absorption of riboflavin (RefSeq, 2002).SLC52A3 is de-regulated in different cancer entities including gastriccancer, esophageal squamous cell carcinoma and cervical cancer. Singlenucleotide polymorphisms of SLC52A3 correlate with cancer risks inesophageal squamous cell carcinoma and gastric cardia adenocarcinomas(Jiang et al., 2014b; Duan et al., 2015; Matnuri et al., 2015; Eli etal., 2012; Aili et al., 2013). Knock-down of SLC52A3 increases p21 andp27 protein levels and decreases their down-stream targets cyclin E1 andCdk2, leading to cell cycle arrest at G1-G1/S. Knock-down of SLC52A3also leads to the activation of caspase-3 and apoptosis (Jiang et al.,2014b).

SLC6A15 encodes a member of the solute carrier family 6 which transportsneutral amino acids. SLC6A15 might play a role in neuronal amino acidtransport and might be associated with major depression (RefSeq, 2002).SLC6A15 is hyper-methylated and thereby down-regulated in colorectalcancer and may be a candidate biomarker for a stool-based assay (Kim etal., 2011b; Mitchell et al., 2014).

SMIM10 (also called CXorf69 or LOC644538) encodes a small integralmembrane protein located on chromosome Xq26.3 (RefSeq, 2002).

SNX14 encodes a member of the sorting nexin family and contains aregulator of G protein signaling (RGS) domain (RefSeq, 2002). SNX14 isdown-regulated upon rasV12/E1A transformation of mouse embryonicfibroblasts and may be associated with tumor development (Vasseur etal., 2005).

SSH1 (also called SSH1L) encodes a member of the slingshot homolog (SSH)family of phosphatases. The SSH family appears to play a role in actindynamics by reactivating cofilin proteins (RefSeq, 2002). SSH1 isover-expressed in pancreatic cancer and associated with tumor cellmigration (Wang et al., 2015k). Inhibition of PKD1 by neuregulin leadsto the localization of SSH1 to F-actin, increased cofilin activity andincreased reorganization of the actin cytoskeleton and cell migration.The SSH1-dependent activation of cofilin is induced by the PI3K/Aktsignaling pathway (Wang et al., 2010; Doppler et al., 2013).

STAT2 operates as a positive regulator in the transcriptional activationresponse elicited by IFNs (Steen and Gamero, 2012). STAT2 may regulatetumor cell response to interferons (Shodeinde et al., 2013). A linkbetween STAT2 and tumorigenesis was observed in transgenic mice lackingSTAT2 (Yue et al., 2015). or expressing constitutively IFN-alpha in thebrain (Wang et al., 2003).

SUPT16H encodes a subunit of FACT (facilitates chromatin transcription),an accessory factor which is needed for the transcription of DNApackaged into chromatin (RefSeq, 2002). SUPT16H is de-regulated inendothelial and stromal components of juvenile nasopharyngealangiofibroma (JNA) and could thereby play a role as a potentialmolecular marker (Silveira et al., 2012). SUPT16H is involved in DNAdouble-strand break repair by remodeling of chromatin. SUPT16H activatesp53 by forming a complex with CK2 (Keller et al., 2001; Kari et al.,2011).

SUSD1 encodes a sushi domain containing protein and is associated withan increased risk of venous thromboembolism (Tang et al., 2013). Theheterozygous SUSD1-ROD1/PTBP3 fusion transcript is expressed in a humanbreast cancer cell line (Newman et al., 2013).

TAF6L encodes a protein with structurally similarity to the histone likeTATA-box binding protein associated factor 6 (TAF6). It is a componentof the PCAF histone acetylase complex which is required for myogenictranscription and differentiation (RefSeq, 2002). The expression ofmiR-145 and miR-196a negatively correlates with the expression of TAF6L(Havelange et al., 2011). TAF6L is inactivated in the small cell lungcancer cell line H187 by forming the fusion transcript TAF6L-GNG3(Fernandez-Cuesta et al., 2015).

TEP1 encodes telomerase associated protein 1, a component of theribonucleoprotein complex responsible for telomerase activity, whichcatalyzes the addition of new telomeres on the chromosome ends (RefSeq,2002; Szaflarski et al., 2011). TEP1 is a main part of vaults to whichalso major vault protein (MVP) belongs (Lara et al., 2011; Mossink etal., 2003). TEP1 is expressed in thyroid carcinoma (Hoang-Vu et al.,2002).

TFPI encodes tissue factor pathway inhibitor, a protease inhibitor thatregulates the tissue factor (TF)-dependent pathway of blood coagulation(RefSeq, 2002). TFPI is expressed in breast cancer, colorectal cancer,and pancreatic cancer cell lines (Kurer, 2007). TFPI induces HIF1alpha,c-Myc, c-SRC, and HDAC2 in breast cancer (Davies et al., 2014). TFPIexpression level is decreased in sarcomas compared to non-malignantlesions (Savitskaya et al., 2012). TFPI inhibits the protease activityof the TF-Vlla complex which is involved in metastasis (Fischer et al.,1999; Sandset and Abildgaard, 1991; Lindahl et al., 1991).

TFPI2 encodes tissue factor pathway inhibitor 2 which can inhibit avariety of serine proteases including factor Vila/tissue factor, factorXa, plasmin, trypsin, chymotrypsin, and plasma kallikrein. This gene hasbeen identified as a tumor suppressor gene in several types of cancer(RefSeq, 2002; Sierko et al., 2007). TFPI2 may be used as a biomarkerfor relapse prediction in pancreatic carcinoma (Zhai et al., 2015c). DNAmethylation of TFPI2 can be used as a biomarker for colorectal cancer ina fecal occult blood test (Koga et al., 2015). TFPI2 induces apoptosisand inhibits invasiveness, growth of neoplasms, metastasis, andangiogenesis (Ghilardi et al., 2015; Amirkhosravi et al., 2007; Sierkoet al., 2007). TFPI2 is hyper-methylated and down-regulated in cancer,and expression is correlated with the degree of cancer, early tumorrecurrence, and poor prognosis (Sun et al., 2016a; Sierko et al., 2007).TFPI2 is down-regulated in pancreatic cancer and cholangiocarcinoma (Chuet al., 2015; Zhai et al., 2015a; Zhai et al., 2015b). TFPI2 ismethylated in gastric cancer, canine diffuse large B-cell lymphoma,acute myeloid leukemia, non-small cell lung cancer, cervical cancer,oral squamous cell carcinoma, inflammation-associated colon cancer, andhepatocellular carcinoma (Qu et al., 2013; Ferraresso et al., 2014; Liuet al., 2014b; Shao et al., 2014; Lai et al., 2014; Hamamoto et al.,2015; Li et al., 2015d; Gerecke et al., 2015; Dong et al., 2015; Sun etal., 2016a). TFPI2 is a well-validated DNA methylation biomarker incancer (Fukushige and Horii, 2013; Huisman et al., 2015).

TGFBI encodes an RGD-containing protein that binds to type I, II and IVcollagens, is induced by transforming growth factor-beta which plays arole in cell-collagen interactions and acts to inhibit cell adhesion(RefSeq, 2002). TGFBI expression was shown to be elevated incholangiocarcinoma, hepatic carcinoma, gastric carcinoma, esophagealsquamous cell carcinoma and clear cell renal cell carcinoma.Furthermore, TGFBI was shown to be associated with colorectal cancer(Lebdai et al., 2015; Ozawa et al., 2014; Zhu et al., 2015a; Han et al.,2015).

TGIF2-C20orf24 encodes a fusion protein that shares sequence identitywith TGIF2 and C20orf24 (RefSeq, 2002).

TMEM154 encodes a transmembrane protein that is associated with anincreased risk for type 2 diabetes and that seems to play a role in betacell function (Harder et al., 2015). TRAM2 encodes translocationassociated membrane protein 2. It is a component of the translocon, agated macromolecular channel that controls the posttranslationalprocessing of nascent secretory and membrane proteins at the endoplasmicreticulum (ER) membrane (RefSeq, 2002). Runx2 may regulate TRAM2expression (Pregizer et al., 2007). SNPs in TRAM2 can increase the riskof bone fracture in ER-positive breast cancer patients (Liu et al.,2014a).

TRPV2 encodes an ion channel that is activated by temperatures above 52degrees Celsius. It may be involved in transduction of high-temperatureheat response s in sensory ganglia (RefSeq, 2002). TRPV2 is de-regulatedin different cancer types including esophageal, prostate, liver andbladder cancer and leukemia. Loss or alterations of TRPV2 lead touncontrolled proliferation and resistance to apoptotic stimuli (Liberatiet al., 2014a; Zhou et al., 2014; Liberati et al., 2014b; Liu et al.,2010a; Morelli et al., 2013). Silencing of TRPV2 in glioma cells leadsto down-regulation of Fas and pro-caspase 8 as well as up-regulation ofCyclin E1, CDK2 E2F1 and Bcl-2-associated X protein. TRPV2over-expression in bladder cancer cells leads to an enhanced cellmigration and invasion (Nabissi et al., 2010; Liu and Wang, 2013).

TSEN15 encodes tRNA splicing endonuclease subunit 15. This endonucleasecatalyzes the removal of introns from tRNA precursors (RefSeq, 2002;Trotta et al., 2006). TSEN15 is a target of miRNA-449a, which functionsas a tumor suppressor in neuroblastoma. TSEN15 plays an important rolein mediating the differentiation-inducing function of miRNA-449a (Zhaoet al., 2015). TSEN15 is associated with cell differentiation potentialin human fetal femur-derived cells (Mirmalek-Sani et al., 2009).

UBE2C (also called UBCH10) encodes a member of the E2ubiquitin-conjugating enzyme family. It is required for the destructionof mitotic cyclins and cell cycle progression (RefSeq, 2002). UBE2C isoften up-regulated by gene amplification, as observed in patients withbreast, lung and colorectal cancer. UBE2C up-regulation correlates withpoor prognosis and tumor progression (Okamoto et al., 2003; Wagner etal., 2004; Fujita et al., 2009; Chen et al., 2010; Hao et al., 2012).UBE2C is up-regulated in U251 glioma cells and in tissues fromcolorectal carcinoma (CRC) patients. UBE2C knock-down induces apoptosisthrough the induction of Bax and p53, down-regulation of Bcl-2 and G2/Marrest of the cell cycle. UBE2C suppression de-regulates cyclin B andERK1 in CRC (Cacciola et al., 2015; Jiang et al., 2010).

UBIAD1 (also called TERE1) encodes a protein containing an UbiAprenyltransferase domain that might be involved in cholesterol andphospholipid metabolism (RefSeq, 2002). The tumor suppressor UBIAD1 isdown-regulated in different cancer entities, including bladder, prostateand renal cancer, and is associated with growth regulation (McGarvey etal., 2001; Fredericks et al., 2011; McGarvey et al., 2003; Fredericks etal., 2013). UBIAD1 regulates the phosphorylation of the growthfactor-related p42/44 MAP kinase. The proper Golgi localization ofUBIAD1 influences its tumor suppressor activities including apoptosis(McGarvey et al., 2005; Wang et al., 2013d).

UBR1 encodes ubiquitin protein ligase E3 component N-recognin 1. Itbinds to a destabilizing N-terminal residue of a substrate protein andparticipates in the formation of a substrate-linked multi-ubiquitinchain, addressing the protein for the proteolytic pathway of theubiquitin system (RefSeq, 2002). Loss or reduction of UBR1 expression isassociated with spontaneous B-cell lymphomas and T-cell acutelymphoblastic leukemia (Chen et al., 2006). UBR1 regulates thehomeostasis of MGMT, a DNA repair enzyme that protects cells fromcarcinogenic effects of alkylating agents (Leng et al., 2015).

UBR2 encodes an E3 ubiquitin ligase of the N-end rule proteolyticpathway that targets proteins with destabilizing N-terminal residues forpolyubiquitylation and proteasome-mediated degradation (RefSeq, 2002).Autoantibodies against UBR2 are detected in serum of patients withautoimmune pancreatitis and pancreatic cancer (Frulloni et al., 2009).UBR2 is up-regulated by tumor cell-induced cachectic stimuli viaactivation of p38beta/MAPK, C/EBPbeta phosphorylation and binding to theUBR2 promotor (Zhang et al., 2013b).

URB1 is required for ribosome biogenesis during early maturation of 60Sribosomal subunits (Rosado and de la Cruz, 2004).

USP11 encodes ubiquitin specific peptidase 11. Protein ubiquitinationcontrols many intracellular processes, including cell cycle progression,transcriptional activation, and signal transduction (RefSeq, 2002).USP11 is a novel regulator of p53, which is required for p53 activationin response to DNA damage (Ke et al., 2014a). USP11 plays a major rolein promyelocytic leukemia and pancreatic cancer (Burkhart et al., 2013;Wu et al., 2014).

USP22 encodes ubiquitin specific peptidase 22 and is located onchromosome 17p11.2 (RefSeq, 2002). High expression of USP22 was observedin hepatocellular carcinoma, colon carcinoma, gastric carcinoma,epithelial ovarian cancer, pancreatic cancer, glioma, salivary adenoidcystic carcinoma, and papillary thyroid carcinoma (Wang et al., 2013b;Dai et al., 2014; Liang et al., 2014a; Liang et al., 2014b; Ji et al.,2015; He et al., 2015; Wang et al., 2015n; Tang et al., 2015). USP22promotes tumor progression and induces epithelial mesenchymal transitionin lung adenocarcinoma (Hu et al., 2015a). USP22 acts as an oncogene byregulating the stability of cyclooxygenase 2 in non-small cell lungcancer (Xiao et al., 2015). USP22 plays a critical regulatory role inthe pathologic processes of nasopharyngeal carcinoma, and it may be apotential treatment target (Zhuang et al., 2015). Over-expression ofUSP22 may contribute to the progression of breast cancer (Zhang et al.,2011).

UTP20 is a component of the U3 small nucleolar RNA protein complex andis involved in 18s rRNA processing (RefSeq, 2002). UTP20 expression isdecreased in metastatic human breast tumor cell lines (Schwirzke et al.,1998; Goodison et al., 2003). UTP20 is expressed at high levels ingastric cancer tissues and premalignant lesions implicating theinvolvement of UTP20 in cell transformation (Xing et al., 2005).

WLS (also called EVI or GPR177) encodes Wntless Wnt ligand secretionmediator. WLS represents an ancient partner for Wnts dedicated topromoting their secretion into the extracellular milieu (Banziger etal., 2006). WLS is over-expressed in different cancer entities includingbreast, gastric, ovarian and colorectal cancer as well as leukemia andis associated with poor outcome (Chiou et al., 2014; Stewart et al.,2015; Lu et al., 2015; Voloshanenko et al., 2013). WLS is important forthe secretion of all Wnt proteins. It regulates the expression ofbeta-catenin and cyclin-D1, thereby influencing cell proliferation (Yanget al., 2015b; Banziger et al., 2006).

YIF1A encodes Yip1 interacting factor homolog A and is located onchromosome 11q13 (RefSeq, 2002). Several mutations (amplifications anddeletions) have been detected in the YIF1A gene in hepatocellularcarcinoma (Nalesnik et al., 2012). YIF1A expression shows a significantdifference between normal and squamous cell carcinoma samples (Sugimotoet al., 2009).

ZRANB3 encodes zinc finger, RAN-binding domain containing 3 and islocated on chromosome 2q21.3 (RefSeq, 2002). ZRANB3 encodes a zincfinger protein that is a structure-specific ATP-dependent endonuclease.It is involved in replication stress response to maintain genomicintegrity (Ciccia et al., 2012; Weston et al., 2012). Single nucleotidepolymorphism rs4954256, located in ZRANB3 on chromosome 2q21.3, wasassociated with a 3.93-fold increase in pathologic complete response toconcurrent chemoradiation therapy in the treatment of esophageal cancer(Chen et al., 2012). ZRANB3 is frequently mutated in endometrial cancer(Lawrence et al., 2014).

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, or 13 amino acids orlonger, and in case of MHC class II peptides (elongated variants of thepeptides of the invention) they can be as long as 14, 15, 16, 17, 18, 19or 20 or more amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences; And R is the number of bases or amino acids in the ReferenceSequence over the length of the alignment with the Compared Sequencewith any gap created in the Reference Sequence also being counted as abase or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity, then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 161 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 161, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) Class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 161. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 161, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gin); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, lie, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) Class-I or—II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) Class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation do not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Preferred variants and motif of the peptides according to SEQ IDNO: 7, 32, 46, and 76. Position 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 7 A LV D I V R S L Variants V I A M V M I M M A A V A I A A A V V V I V V A TV T I T T A Q V Q I Q Q A SEQ ID NO. 32 Y V D D G L I S L Variants I V II I I A M V M I M M A A V A I A A A L V L I L L A T V T I T T A Q V Q IQ Q A SEQ ID NO. 46 T M V E H N Y Y V Variants L I A A L A I A A A L L LI L L A V L V I V V A T L T I T T A Q L Q I Q Q A SEQ ID NO. 76 L V S ES S D V L P K L V L I L L L A M V M I M L M A A V A I A L A A V I L A TV T I T L T A Q V Q I Q L Q A

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 μM, andmost preferably no more than about 10 μM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 161.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 161 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “li”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide can be used to form intra-molecularcrosslinks between a lysine residue and a glutamic acid residue. Forexample, diethylpyrocarbonate is a reagent for the modification ofhistidyl residues in proteins. Histidine can also be modified using4-hydroxy-2-nonenal. The reaction of lysine residues and other α-aminogroups is, for example, useful in binding of peptides to surfaces or thecross-linking of proteins/peptides. Lysine is the site of attachment ofpoly(ethylene)glycol and the major site of modification in theglycosylation of proteins. Methionine residues in proteins can bemodified with e.g. iodoacetamide, bromoethylamine, and chloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of TUMAPs recordedfrom pancreatic cancer samples (N=20 A*02-positive samples) with thefragmentation patterns of corresponding synthetic reference peptides ofidentical sequences. Since the peptides were directly identified asligands of HLA molecules of tumor cells, these results provide directevidence for the processing and presentation of the identified peptideson pancreatic cancer.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from pancreatic cancer samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on pancreatic cancer samples confirmingtheir presentation on pancreatic cancer.

TUMAPs identified on multiple pancreatic cancer and normal tissues werequantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably pancreatic cancer, that over- or exclusivelypresent the peptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on humanpancreatic cancer samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy pancreatic cells or other normal tissue cells, demonstrating ahigh degree of tumor association of the source genes (see Example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean apancreatic cancer sample, but not on normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. pancreatic cancer cells presenting the derivedpeptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are HAVCR1-001 peptides capable of binding to TCRs andantibodies when presented by an MHC molecule. The present descriptionalso relates to nucleic acids, vectors and host cells for expressingTCRs and peptides of the present description; and methods of using thesame.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to an HAVCR1-001peptide-HLA molecule complex with a binding affinity (KD) of about 100μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM orless. More preferred are high affinity TCRs having binding affinities ofabout 1 μM or less, about 100 nM or less, about 50 nM or less, about 25nM or less. Non-limiting examples of preferred binding affinity rangesfor TCRs of the present invention include about 1 nM to about 10 nM;about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM toabout 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM;about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM toabout 90 nM; and about 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for an HAVCR1-001 peptide-HLAmolecule complex of 100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a HAVCR1-001 peptide-HLA molecule complex, whichis at least double that of a TCR comprising the unmutated TCR alphachain and/or unmutated TCR beta chain. Affinity-enhancement oftumor-specific TCRs, and its exploitation, relies on the existence of awindow for optimal TCR affinities. The existence of such a window isbased on observations that TCRs specific for HLA-A2-restricted pathogenshave KD values that are generally about 10-fold lower when compared toTCRs specific for HLA-A2-restricted tumor-associated self-antigens. Itis now known, although tumor antigens have the potential to beimmunogenic, because tumors arise from the individual's own cells onlymutated proteins or proteins with altered translational processing willbe seen as foreign by the immune system. Antigens that are upregulatedor overexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to HAVCR1-001 can be enhanced by methods well known in theart.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/HAVCR1-001 monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRap geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with HAVCR1-001, incubating PBMCs obtained from the transgenicmice with tetramer-phycoerythrin (PE), and isolating the high avidityT-cells by fluorescence activated cell sorting (FACS)-Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 161, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand Helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF—), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, Bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets.

The composition is designed in such a way that each tumor is expected toexpress several of the antigens and cover several independent pathwaysnecessary for tumor growth and maintenance. Thus, the vaccine can easilybe used “off-the-shelf” for a larger patient population. This means thata pre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one Ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed, aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 161,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 161, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 161 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:161 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 161, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) Class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 161.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of pancreatic cancer.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 161 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are pancreatic cancer cells orother solid or hematological tumor cells such as lung cancer, kidneycancer, brain cancer, stomach cancer, colon or rectal cancer, livercancer, prostate cancer, leukemia, breast cancer, Merkel cell carcinoma(MCC), melanoma, ovarian cancer, esophageal cancer, urinary bladdercancer, endometrial cancer, gall bladder cancer, and bile duct cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of pancreatic cancer. The present invention also relates tothe use of these novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a pancreatic cancer marker(poly)peptide, delivery of a toxin to a pancreatic cancer cellexpressing a cancer marker gene at an increased level, and/or inhibitingthe activity of a pancreatic cancer marker polypeptide) according to theinvention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length pancreatic cancer marker polypeptides orfragments thereof may be used to generate the antibodies of theinvention. A polypeptide to be used for generating an antibody of theinvention may be partially or fully purified from a natural source, ormay be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 161polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the pancreatic cancer markerpolypeptide used to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating pancreaticcancer, the efficacy of the therapeutic antibody can be assessed invarious ways well known to the skilled practitioner. For instance, thesize, number, and/or distribution of cancer in a subject receivingtreatment may be monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occur in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 161, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPCs-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition, plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 161.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) A container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) Optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) Optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from pancreaticcancer, the medicament of the invention is preferably used to treatpancreatic cancer.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue ofpancreatic cancer patients with various HLA-A HLA-B and HLA-C alleles.It may contain MHC class I and MHC class II peptides or elongated MHCclass I peptides. In addition to the tumor associated peptides collectedfrom several pancreatic cancer samples, the warehouse may containHLA-A*02 and HLA-A*24 marker peptides. These peptides allow comparisonof the magnitude of T-cell immunity induced by TUMAPS in a quantitativemanner and hence allow important conclusion to be drawn on the capacityof the vaccine to elicit anti-tumor responses. Secondly, they functionas important positive control peptides derived from a “non-self” antigenin the case that any vaccine-induced T-cell responses to TUMAPs derivedfrom “self” antigens in a patient are not observed. And thirdly, it mayallow conclusions to be drawn, regarding the status of immunocompetenceof the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, pancreatic cancer samples andblood from healthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue(pancreatic cancer) compared with a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as frompancreatic cancer patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising:

(a) identifying tumor-associated peptides (TUMAPs) presented by a tumorsample from the individual patient; (b) comparing the peptidesidentified in (a) with a warehouse (database) of peptides as describedabove; and (c) selecting at least one peptide from the warehouse(database) that correlates with a tumor-associated peptide identified inthe patient. For example, the TUMAPs presented by the tumor sample areidentified by:

(a1) comparing expression data from the tumor sample to expression datafrom a sample of normal tissue corresponding to the tissue type of thetumor sample to identify proteins that are over-expressed or aberrantlyexpressed in the tumor sample; and (a2) correlating the expression datawith sequences of MHC ligands bound to MHC class I and/or class IImolecules in the tumor sample to identify MHC ligands derived fromproteins over-expressed or aberrantly expressed by the tumor.Preferably, the sequences of MHC ligands are identified by eluting boundpeptides from MHC molecules isolated from the tumor sample, andsequencing the eluted ligands. Preferably, the tumor sample and thenormal tissue are obtained from the same patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (al) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from pancreatic cancer samples and since it wasdetermined that these peptides are not or at lower levels present innormal tissues, these peptides can be used to diagnose the presence of acancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for pancreatic cancer. Presence of groups of peptides canenable classification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGURES

FIGS. 1A to 1AF show the over-presentation of various peptides in normaltissues (white bars) and pancreatic cancer (black bars). FIG. 1A) Genesymbol(s): PTGS1, PTGS2, Peptide: ILIGETIKI (SEQ ID NO.: 3), Tissuesfrom left to right: 1 adipose tissue, 3 adrenal glands, 6 arteries, 5bone marrows, 7 brains, 3 breasts, 1 nerve, 13 colons, 1 ovary, 8esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 21 livers, 46 lungs, 3lymph nodes, 4 leukocyte samples, 3 ovaries, 4 peripheral nerves, 1peritoneum, 3 pituitary glands, 2 placentas, 3 pleuras, 3 prostates, 6recti, 7 salivary glands, 3 skeletal muscles, 5 skins, 2 smallintestines, 4 spleens, 7 stomachs, 4 testes, 3 thymi, 4 thyroid glands,7 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 2 veins, 7 pancreas,20 pancreatic cancer cell line and xenograft samples. The peptide hasadditionally been detected on 4/91 lung cancers, 1/20 ovarian cancers,1/24 colorectal cancers, 1/18 kidney cancers, and 1/4 urinary bladdercancers (not shown). FIG. 1B) Gene symbol(s): COL1A2, Peptide: FVDTRTLL(SEQ ID NO.: 1), Tissues from left to right: 1 adipose tissue, 3 adrenalglands, 6 arteries, 5 bone marrows, 7 brains, 3 breasts, 1 nerve, 13colons, 1 ovary, 8 esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 21livers, 46 lungs, 3 lymph nodes, 4 leukocyte samples, 3 ovaries, 4peripheral nerves, 1 peritoneum, 3 pituitary glands, 2 placentas, 3pleuras, 3 prostates, 6 recti, 7 salivary glands, 3 skeletal muscles, 5skins, 2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymi, 4thyroid glands, 7 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 2veins, 7 pancreas, 20 pancreatic cancer cell line and xenograft samples.The peptide has additionally been detected on 3/91 lung cancers and 1/17esophageal cancers. FIG. 1C) Gene symbol(s): PTPN14, Peptide: AQYKFVYQV(SEQ ID NO.: 12), Tissues from left to right: 1 adipose tissue, 3adrenal glands, 6 arteries, 5 bone marrows, 7 brains, 3 breasts, 1nerve, 13 colons, 1 ovary, 8 esophagi, 2 gallbladders, 5 hearts, 16kidneys, 21 livers, 46 lungs, 3 lymph nodes, 4 leukocyte samples, 3ovaries, 4 peripheral nerves, 1 peritoneum, 3 pituitary glands, 2placentas, 3 pleuras, 3 prostates, 6 recti, 7 salivary glands, 3skeletal muscles, 5 skins, 2 small intestines, 4 spleens, 7 stomachs, 4testes, 3 thymi, 4 thyroid glands, 7 tracheas, 3 ureters, 6 urinarybladders, 2 uteri, 2 veins, 7 pancreas, 20 pancreatic cancer cell lineand xenograft samples. The peptide has additionally been detected on1/20 ovarian cancers, 2/17 esophageal cancers, 1/46 stomach cancers,1/91 lung cancers, and 1/18 kidney cancers. FIG. 1D) Gene symbol(s):UBR1, Peptide: SLMDPNKFLLL (SEQ ID NO.: 115), Tissues from left toright: 13 pancreatic cell lines, 2 PBMC cultures, 1 prostate cellculture, 3 skin cell lines, 7 normal tissues (1 liver, 2 lungs, 2spleens, 1 stomach, 1 trachea), 62 cancer tissues (8 brain cancers, 2breast cancers, 2 colon cancers, 1 esophageal cancer, 1 gallbladdercancer, 5 kidney cancers, 3 leukemias, 6 liver cancers, 19 lung cancers,5 ovarian cancers, 1 pancreatic cancer, 3 prostate cancers, 3 rectalcancers, 1 skin cancer, 2 urinary bladder cancers). The normal tissuepanel (no disease) and the cancer cell lines and xenografts tested werethe same as in FIG. 1A-1C, consisting of 1 adipose tissue, 3 adrenalglands, 6 arteries, 5 bone marrows, 7 brains, 3 breasts, 1 nerve, 13colons, 1 ovary, 8 esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 21livers, 46 lungs, 3 lymph nodes, 4 leukocyte samples, 3 ovaries, 4peripheral nerves, 1 peritoneum, 3 pituitary glands, 2 placentas, 3pleuras, 3 prostates, 6 recti, 7 salivary glands, 3 skeletal muscles, 5skins, 2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymi, 4thyroid glands, 7 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 2veins, 7 pancreas, 20 pancreatic cancer cell line and xenograft samples.The peptide has additionally been detected on 1/6 breast cancers, 5/24colorectal cancers, 1/2 gallbladder/bile duct cancers, 6/16 livercancers, 1/2 melanomas, 5/20 ovarian cancers, 1/17 esophageal cancers,3/12 leukemias, 7/29 brain cancers, 16/91 non-small cell lungcarcinomas, 3/33 prostate cancers, 3/18 kidney cancers, 3/14 small celllung carcinomas, and 1/4 urinary bladder cancers. Discrepanciesregarding the list of tumor types between FIG. 1D and table 4 may be dueto the more stringent selection criteria applied in table 4 (for detailsplease refer to table 4). FIG. 1D shows all samples with detectablepresentation of the peptide Y, regardless of over-presentationparameters and technical sample quality test. FIG. 1E) Gene symbol(s):NUP205, Peptide: ALLTGIISKA (SEQ ID NO.: 5), Tissues from left to right:6 adipose tissues, 8 adrenal glands, 24 blood cells, 15 blood vessels,10 bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes, 3gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23 livers, 49lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6 parathyroid glands, 1peritoneum, 6 pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7salivary glands, 10 skeletal muscles, 11 skins, 8 small intestines, 12spleens, 7 stomachs, 5 testes, 3 thymi, 3 thyroid glands, 15 tracheas, 7ureters, 8 urinary bladders, 6 uteri, 10 pancreases, 20 pancreaticcancer cell line and xenograft samples. The peptide has additionallybeen found on 2/34 brain cancers, 1/18 breast cancers, 2/29 colon orrectum cancers, 1/18 esophageal cancers, 1/8 head and neck cancers, 1/21liver cancers, 8/107 lung cancers, 1/20 lymph node cancers, 1/20 ovariancancers, 1/18 skin cancers, 2/15 urinary bladder cancers, 1/16 uteruscancers. FIG. 1F) Gene symbol(s): NUP160, Peptide: ALWHDAENQTW (SEQ IDNO.: 19), Tissues from left to right: 6 adipose tissues, 8 adrenalglands, 24 blood cells, 15 blood vessels, 10 bone marrows, 14 brains, 7breasts, 9 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23large intestines, 23 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2ovaries, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands, 7placentas, 1 pleura, 3 prostates, 7 salivary glands, 10 skeletalmuscles, 11 skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes,3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary bladders, 6uteri, 10 pancreases, 20 pancreatic cancer cell line and xenograftsamples. The peptide has additionally been found on 2/17 gallbladder orbile duct cancers, 2/34 brain cancers, 1/18 breast cancers, 1/18esophageal cancers, 1/21 liver cancers, 8/107 lung cancers, 2/18 skincancers, 2/15 urinary bladder cancers, 1/16 uterus cancers. FIG. 1G)Gene symbol(s): C11 orf80, Peptide: ILSTEIFGV (SEQ ID NO.: 22), Tissuesfrom left to right: 6 adipose tissues, 8 adrenal glands, 24 blood cells,15 blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9 esophagi, 2eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6 parathyroidglands, 1 peritoneum, 6 pituitary glands, 7 placentas, 1 pleura, 3prostates, 7 salivary glands, 10 skeletal muscles, 11 skins, 8 smallintestines, 12 spleens, 7 stomachs, 5 testes, 3 thymi, 3 thyroid glands,15 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 10 pancreases, 20pancreatic cancer cell line and xenograft samples. The peptide hasadditionally been found on 3/18 breast cancers, 1/17 gallbladdercancers, 1/8 head and neck cancers, 5/17 leukocytic leukemia cancers,6/107 lung cancers, 4/20 lymph node cancers, 1/20 ovarian cancers, 1/19pancreas cancers, 1/18 skin cancers, 1/21 stomach cancers. FIG. 1H) Genesymbol(s): FAM83D, Peptide: FLNPDEVHAI (SEQ ID NO.: 37), Tissues fromleft to right: 6 adipose tissues, 8 adrenal glands, 24 blood cells, 15blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9 esophagi, 2eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6 parathyroidglands, 1 peritoneum, 6 pituitary glands, 7 placentas, 1 pleura, 3prostates, 7 salivary glands, 10 skeletal muscles, 11 skins, 8 smallintestines, 12 spleens, 7 stomachs, 5 testes, 3 thymi, 3 thyroid glands,15 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 10 pancreases, 20pancreatic cancer cell line and xenograft samples. The peptide hasadditionally been found on 2/17 gallbladder or bile duct cancers, 2/34brain cancers, 3/18 breast cancers, 6/29 colon or rectum cancers, 2/18esophageal cancers, 2/8 head and neck cancers, 1/23 kidney cancers, 5/21liver cancers, 25/107 lung cancers, 4/20 lymph node cancers, 7/20ovarian cancers, 1/87 prostate cancers, 2/18 skin cancers, 2/45 stomachcancers, 6/15 urinary bladder cancers, 3/16 uterus cancers. FIG. 1I)Gene symbol(s): DCBLD2, Peptide: TMVEHNYYV (SEQ ID NO.: 46), Tissuesfrom left to right: 6 adipose tissues, 8 adrenal glands, 24 blood cells,15 blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9 esophagi, 2eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6 parathyroidglands, 1 peritoneum, 6 pituitary glands, 7 placentas, 1 pleura, 3prostates, 7 salivary glands, 10 skeletal muscles, 11 skins, 8 smallintestines, 12 spleens, 7 stomachs, 5 testes, 3 thymi, 3 thyroid glands,15 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 10 pancreases, 20pancreatic cancer cell line and xenograft samples. The peptide hasadditionally been found on 1/18 esophageal cancer, 1/17 gallbladdercancers, 1/8 head and neck cancers, 3/23 kidney cancers, 9/107 lungcancers, 7/20 ovarian cancers, 1/19 pancreas cancers, 1/18 skin cancers,1/45 stomach cancers, 2/15 urinary bladder cancers, 1/16 uterus cancers.FIG. 1J) Gene symbol(s): SHCBP1, Peptide: RLSELGITQA (SEQ ID NO.: 57),Tissues from left to right: 6 adipose tissues, 8 adrenal glands, 24blood cells, 15 blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 largeintestines, 23 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas, 1pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11 skins, 8small intestines, 12 spleens, 7 stomachs, 5 testes, 3 thymi, 3 thyroidglands, 15 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 10pancreases, 20 pancreatic cancer cell line and xenograft samples. Thepeptide has additionally been found on 1/34 brain cancers, 1/18 breastcancers, 2/18 esophageal cancers, 2/8 head and neck cancers, 1/21 livercancers, 8/107 lung cancers, 4/20 lymph node cancers, 1/18 myeloid cellcancers, 4/20 ovarian cancers, 4/18 skin cancers, 2/15 urinary bladdercancers, 1/16 uterus cancers. FIG. 1K) Gene symbol(s): CTHRC1, Peptide:VLFSGSLRL (SEQ ID NO.: 69), Tissues from left to right: 6 adiposetissues, 8 adrenal glands, 24 blood cells, 15 blood vessels, 10 bonemarrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes, 3 gallbladders, 16hearts, 17 kidneys, 23 large intestines, 23 livers, 49 lungs, 7 lymphnodes, 12 nerves, 2 ovaries, 6 parathyroid glands, 1 peritoneum, 6pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands,10 skeletal muscles, 11 skins, 8 small intestines, 12 spleens, 7stomachs, 5 testes, 3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8urinary bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell lineand xenograft samples. The peptide has additionally been found on 2/18breast cancers, 1/18 esophageal cancers, 1/17 gallbladder cancers, 9/107lung cancers, 1/20 ovarian cancers. FIG. 1L) Gene symbol(s): CDC27,Peptide: KISTITPQI (SEQ ID NO.: 123), Tissues from left to right: 6adipose tissues, 8 adrenal glands, 24 blood cells, 15 blood vessels, 10bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes, 3 gallbladders,16 hearts, 17 kidneys, 23 large intestines, 23 livers, 49 lungs, 7 lymphnodes, 12 nerves, 2 ovaries, 6 parathyroid glands, 1 peritoneum, 6pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands,10 skeletal muscles, 11 skins, 8 small intestines, 12 spleens, 7stomachs, 5 testes, 3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8urinary bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell lineand xenograft samples. The peptide has additionally been found on 2/34brain cancers, 2/8 head and neck cancers, 1/23 kidney cancers, 1/17leukocytic leukemia cancers, 2/21 liver cancers, 7/107 lung cancers,2/20 lymph node cancers, 1/18 myeloid cell cancers, 1/18 skin cancers,1/45 stomach cancers, 2/15 urinary bladder cancers, 3/16 uterus cancers.FIG. 1M) Gene symbol(s): UBE2C, Peptide: ALYDVRTILL (SEQ ID NO.: 128),Tissues from left to right: 6 adipose tissues, 8 adrenal glands, 24blood cells, 15 blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 largeintestines, 23 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas, 1pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11 skins, 8small intestines, 12 spleens, 7 stomachs, 5 testes, 3 thymi, 3 thyroidglands, 15 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 10pancreases, 20 pancreatic cancer cell line and xenograft samples. Thepeptide has additionally been found on 2/18 breast cancers, 3/29 colonor rectum cancers, 1/17 leukocytic leukemia cancers, 8/107 lung cancers,1/20 lymph node cancers, 1/20 ovarian cancers, 1/15 urinary bladdercancers. FIG. 1N) Gene symbol(s): MBTPS2, Peptide: VLISGVVHEI (SEQ IDNO.: 146), Tissues from left to right: 6 adipose tissues, 8 adrenalglands, 24 blood cells, 15 blood vessels, 10 bone marrows, 14 brains, 7breasts, 9 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23large intestines, 23 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2ovaries, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands, 7placentas, 1 pleura, 3 prostates, 7 salivary glands, 10 skeletalmuscles, 11 skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes,3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary bladders, 6uteri, 10 pancreases, 20 pancreatic cancer cell line and xenograftsamples. The peptide has additionally been found on 7/34 brain cancers,1/18 breast cancers, 2/29 colon or rectum cancers, 1/18 esophagealcancers, 1/23 kidney cancers, 3/21 liver cancers, 5/107 lung cancers,1/20 lymph node cancers, 2/20 ovarian cancers, 1/87 prostate cancers,3/18 skin cancers, 1/16 uterus cancers. FIG. 1O) Gene symbol(s): PFDN1,Peptide: KLADIQIEQL (SEQ ID NO.: 89), Tissues from left to right: 6adipose tissues, 8 adrenal glands, 24 blood cells, 15 blood vessels, 10bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes, 3 gallbladders,16 hearts, 17 kidneys, 23 large intestines, 23 livers, 49 lungs, 7 lymphnodes, 12 nerves, 2 ovaries, 6 parathyroid glands, 1 peritoneum, 6pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands,10 skeletal muscles, 11 skins, 8 small intestines, 12 spleens, 7stomachs, 5 testes, 3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8urinary bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell lineand xenograft samples. The peptide has additionally been found on 2/29colon or rectum cancers, 1/17 leukocytic leukemia cancers, 4/107 lungcancers, 4/20 ovarian cancers, 4/16 urinary bladder cancers. FIG. 1P)Gene symbol(s): PKP3, Peptide: ALVEENGIFEL (SEQ ID NO.: 101), Tissuesfrom left to right: 6 adipose tissues, 8 adrenal glands, 24 blood cells,15 blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9 esophagi, 2eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6 parathyroidglands, 1 peritoneum, 6 pituitary glands, 7 placentas, 1 pleura, 3prostates, 7 salivary glands, 10 skeletal muscles, 11 skins, 8 smallintestines, 12 spleens, 7 stomachs, 5 testes, 3 thymi, 3 thyroid glands,15 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 10 pancreases, 20pancreatic cancer cell line and xenograft samples. The peptide hasadditionally been found on 1/17 bile duct cancers, 2/18 breast cancers,2/29 colon or rectum cancers, 2/18 esophageal cancers, 2/8 head and neckcancers, 1/21 liver cancers, 7/107 lung cancers, 6/20 ovarian cancers,3/87 prostate cancers, 4/15 urinary bladder cancers, 1/16 uteruscancers. FIG. 1Q) Gene symbol(s): GFPT2, Peptide: LMMSEDRISL (SEQ IDNO.: 113), Tissues from left to right: 6 adipose tissues, 8 adrenalglands, 24 blood cells, 15 blood vessels, 10 bone marrows, 14 brains, 7breasts, 9 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23large intestines, 23 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2ovaries, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands, 7placentas, 1 pleura, 3 prostates, 7 salivary glands, 10 skeletalmuscles, 11 skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes,3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary bladders, 6uteri, 10 pancreases, 20 pancreatic cancer cell line and xenograftsamples. The peptide has additionally been found on 3/17 gallbladder orbile duct cancers, 5/34 brain cancers, 3/18 breast cancers, 2/29 colonor rectum cancers, 2/18 esophageal cancers, 1/8 head and neck cancers,1/21 liver cancers, 18/107 lung cancers, 3/20 lymph node cancers, 1/19pancreas cancers, 1/87 prostate cancers, 2/18 skin cancers, 2/15 urinarybladder cancers, 1/16 uterus cancers. FIG. 1R) Gene symbol(s): CCT4,Peptide: ALSDLALHFL (SEQ ID NO.: 127), Tissues from left to right: 6adipose tissues, 8 adrenal glands, 24 blood cells, 15 blood vessels, 10bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes, 3 gallbladders,16 hearts, 17 kidneys, 23 large intestines, 23 livers, 49 lungs, 7 lymphnodes, 12 nerves, 2 ovaries, 6 parathyroid glands, 1 peritoneum, 6pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands,10 skeletal muscles, 11 skins, 8 small intestines, 12 spleens, 7stomachs, 5 testes, 3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8urinary bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell lineand xenograft samples. The peptide has additionally been found on 1/34brain cancers, 2/18 breast cancers, 2/8 head and neck cancers, 3/17leukocytic leukemia cancers, 1/21 liver cancers, 3/107 lung cancers,4/20 lymph node cancers, 2/18 myeloid cell cancers, 1/20 ovariancancers, 3/18 skin cancers, 4/15 urinary bladder cancers. FIG. 1S) Genesymbol(s): NUP205, Peptide: ALLTGIISKA (SEQ ID NO.: 5), Tissues fromleft to right: 12 cancer cell lines, 1 normal tissue (1 spleen), 22cancer tissues (2 brain cancers, 1 breast cancer, 1 colon cancer, 1esophageal cancer, 1 head and neck cancer, 1 liver cancer, 8 lungcancers, 1 lymph node cancer, 1 ovarian cancer, 1 rectum cancer, 1 skincancer, 2 urinary bladder cancers, 1 uterus cancer). The normal tissuepanel tested was the same as in FIG. 1E-1R. FIG. 1T) Gene symbol(s):NUP160, Peptide: ALWHDAENQTVV (SEQ ID NO.: 19), Tissues from left toright: 13 cancer cell lines, 1 primary culture, 1 normal tissue (1spleen), 20 cancer tissues (1 bile duct cancer, 2 brain cancers, 1breast cancer, 1 esophageal cancer, 1 gallbladder cancer, 1 livercancer, 8 lung cancers, 2 skin cancers, 2 urinary bladder cancers, 1uterus cancer). The normal tissue panel tested was the same as in FIG.1E-1R. FIG. 1U) Gene symbol(s): C11 orf80, Peptide: ILSTEIFGV (SEQ IDNO.: 22), Tissues from left to right: 1 cancer cell line, 3 primarycultures, 1 normal tissue (1 lymph node), 24 cancer tissues (3 breastcancers, 1 gallbladder cancer, 1 head and neck cancer, 5 leukocyticleukemia cancers, 6 lung cancers, 4 lymph node cancers, 1 ovariancancer, 1 pancreas cancer, 1 skin cancer, 1 stomach cancer). The normaltissue panel tested was the same as in FIG. 1E-1R. FIG. 1V) Genesymbol(s): FAM83D, Peptide: FLNPDEVHAI (SEQ ID NO.: 37), Tissues fromleft to right: 16 cancer cell lines, 3 primary cultures, 1 normal tissue(1 trachea), 73 cancer tissues (1 bile duct cancer, 2 brain cancers, 3breast cancers, 4 colon cancers, 2 esophageal cancers, 1 gallbladdercancer, 2 head and neck cancers, 1 kidney cancer, 5 liver cancers, 25lung cancers, 4 lymph node cancers, 7 ovarian cancers, 1 prostatecancer, 2 rectum cancers, 2 skin cancers, 2 stomach cancers, 6 urinarybladder cancers, 3 uterus cancers). The normal tissue panel tested wasthe same as in FIG. 1E-1R. FIG. 1W) Gene symbol(s): DCBLD2, Peptide:TMVEHNYYV (SEQ ID NO.: 46), Tissues from left to right: 4 cancer celllines, 1 primary culture, 28 cancer tissues (1 esophageal cancer, 1gallbladder cancer, 1 head and neck cancer, 3 kidney cancers, 9 lungcancers, 7 ovarian cancers, 1 pancreas cancer, 1 skin cancer, 1 stomachcancer, 2 urinary bladder cancers, 1 uterus cancer). The normal tissuepanel tested was the same as in FIG. 1E-1R. FIG. 1X) Gene symbol(s):SHCBP1, Peptide: RLSELGITQA (SEQ ID NO.: 57), Tissues from left toright: 20 cancer cell lines, 2 primary cultures, 2 normal tissues (1bone marrow, 1 placenta), 31 cancer tissues (1 brain cancer, 1 breastcancer, 2 esophageal cancers, 2 head and neck cancers, 1 liver cancer, 8lung cancers, 4 lymph node cancers, 1 myeloid cell cancer, 4 ovariancancers, 4 skin cancers, 2 urinary bladder cancers, 1 uterus cancer).The normal tissue panel tested was the same as in FIG. 1E-1R. FIG. 1Y)Gene symbol(s): CTHRC1, Peptide: VLFSGSLRL (SEQ ID NO.: 69), Tissuesfrom left to right: 5 cancer cell lines, 14 cancer tissues (2 breastcancers, 1 esophageal cancer, 1 gallbladder cancer, 9 lung cancers, 1ovarian cancer). The normal tissue panel tested was the same as in FIG.1E-1R. FIG. 1Z) Gene symbol(s): CDC27, Peptide: KISTITPQI (SEQ ID NO.:123), Tissues from left to right: 19 cancer cell lines, 2 primarycultures, 3 normal tissues (1 adrenal gland, 1 liver, 1 placenta), 25cancer tissues (2 brain cancers, 2 head and neck cancers, 1 kidneycancer, 1 leukocytic leukemia cancer, 2 liver cancers, 7 lung cancers, 2lymph node cancers, 1 myeloid cell cancer, 1 skin cancer, 1 stomachcancer, 2 urinary bladder cancers, 3 uterus cancers). The normal tissuepanel tested was the same as in FIG. 1E-1R. FIG. 1AA) Gene symbol(s):UBE2C, Peptide: ALYDVRTILL (SEQ ID NO.: 128), Tissues from left toright: 10 cancer cell lines, 17 cancer tissues (2 breast cancers, 1cecum cancer, 2 colon cancers, 1 leukocytic leukemia cancer, 8 lungcancers, 1 lymph node cancer, 1 ovarian cancer, 1 urinary bladdercancer).

The normal tissue panel tested was the same as in FIG. 1E-1R. FIG. 1AB)Gene symbol(s): MBTPS2, Peptide: VLISGVVHEI (SEQ ID NO.: 146), Tissuesfrom left to right: 16 cancer cell lines, 2 primary cultures, 2 normaltissues (1 spleen, 1 uterus), 28 cancer tissues (7 brain cancers, 1breast cancer, 2 colon cancers, 1 esophageal cancer, 1 kidney cancer, 3liver cancers, 5 lung cancers, 1 lymph node cancer, 2 ovarian cancers, 1prostate cancer, 3 skin cancers, 1 uterus cancer). The normal tissuepanel tested was the same as in FIG. 1E-1R. FIG. 1AC) Gene symbol(s):PFDN1, Peptide: KLADIQIEQL (SEQ ID NO.: 89), Tissues from left to right:11 cancer cell lines, 2 normal tissues (2 adrenal glands), 15 cancertissues (2 colon cancers, 1 leukocytic leukemia cancer, 4 lung cancers,4 ovarian cancers, 4 urinary bladder cancers). The normal tissue paneltested was the same as in FIG. 1E-1R. FIG. 1AD) Gene symbol(s): PKP3,Peptide: ALVEENGIFEL (SEQ ID NO.: 101), Tissues from left to right: 3cancer cell lines, 3 primary cultures, 2 normal tissues (2 colons), 31cancer tissues (1 bile duct cancer, 2 breast cancers, 1 cecum cancer, 1colon cancer, 2 esophageal cancers, 2 head and neck cancers, 1 livercancer, 7 lung cancers, 6 ovarian cancers, 3 prostate cancers, 4 urinarybladder cancers, 1 uterus cancer). The normal tissue panel tested wasthe same as in FIG. 1E-1R. FIG. 1AE) Gene symbol(s): GFPT2, Peptide:LMMSEDRISL (SEQ ID NO.: 113), Tissues from left to right: 8 cancer celllines, 1 normal tissue (1 eye), 45 cancer tissues (1 bile duct cancer, 5brain cancers, 3 breast cancers, 1 colon cancer, 2 esophageal cancers, 2gallbladder cancers, 1 head and neck cancer, 1 liver cancer, 18 lungcancers, 3 lymph node cancers, 1 pancreas cancer, 1 prostate cancer, 1rectum cancer, 2 skin cancers, 2 urinary bladder cancers, 1 uteruscancer). The normal tissue panel tested was the same as in FIG. 1E-1R.FIG. 1AF) Gene symbol(s): CCT4, Peptide: ALSDLALHFL (SEQ ID NO.: 127),Tissues from left to right: 9 cancer cell lines, 26 cancer tissues (1bone marrow cancer, 1 brain cancer, 2 breast cancers, 2 head and neckcancers, 3 leukocytic leukemia cancers, 1 liver cancer, 3 lung cancers,4 lymph node cancers, 1 myeloid cell cancer, 1 ovarian cancer, 3 skincancers, 4 urinary bladder cancers). The normal tissue panel tested wasthe same as in FIG. 1E-1R.

FIGS. 2A to 2C show exemplary expression profiles (relative expressioncompared to normal pancreas) of source genes of the present inventionthat are highly over-expressed or exclusively expressed in pancreaticcancer in a panel of normal tissues (white bars) and 9 pancreatic cancersamples (black bars). Tissues from left to right: adrenal gland, artery,bone marrow, brain (whole), breast, colon, esophagus, heart, kidney(triplicate), leukocytes, liver, lung, lymph node, ovary, pancreas,placenta, prostate, salivary gland, skeletal muscle, skin, smallintestine, spleen, stomach, testis, thymus, thyroid gland, urinarybladder, uterine cervix, uterus, vein, 9 pancreatic cancer samples. FIG.2A) SHCBP1; FIG. 2B) FN1; and FIG. 2C) PLEC.

FIGS. 3A to 3D show exemplary immunogenicity data: flow cytometryresults after peptide-specific multimer staining. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 incomplex with SeqID No 125 peptide (FIG. 3A, left panel), SeqID No 148peptide (FIG. 3B, left panel), SeqID No 156 peptide (FIG. 3C, leftpanel), SeqID No 178 peptide (FIG. 3D, left panel, top), and SeqID No177 peptide (FIG. 3D, left panel, bottom), respectively. After threecycles of stimulation, the detection of peptide-reactive cells wasperformed by 2D multimer staining with A*02/SeqID No 125 (FIG.3A),A*02/SeqID No 148 (FIG. 3B) or A*02/SeqID No 156 (FIG. 3C). Rightpanels (FIGS. 3A, 3B, 3C, and 3D) show control staining of cellsstimulated with irrelevant A*02/peptide complexes. Viable singlet cellswere gated for CD8+ lymphocytes. Boolean gates helped excludingfalse-positive events detected with multimers specific for differentpeptides. Frequencies of specific multimer+ cells among CD8+ lymphocytesare indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues and cell lines were obtained from UniversityHospital of Tubingen, Germany, University Hospital of Heidelberg,Germany, NMI Reutlingen, Germany, Md. Anderson Cancer Center, Houston,Tex., USA. Normal tissues were obtained from Asterand, Detroit, USA andRoyston, Herts, UK; Bio-Options Inc., CA, USA; BioServe, Beltsville,Md., USA; Capital BioScience Inc., Rockville, Md., USA; Geneticist Inc.,Glendale, CA, USA; Tissue Solutions Ltd, Glasgow, Scotland, UK;University Hospital of Geneva; University Hospital of Heidelberg; KyotoPrefectural University of Medicine (KPUM); University Hospital Munich;ProteoGenex Inc., Culver City, Calif., USA; University Hospital ofTübingen, Germany. Written informed consents of all donors had beengiven before surgery or autopsy. Tissues were shock-frozen immediatelyafter excision and stored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from frozen tissue samples were obtained by immuneprecipitation according to a slightly modified protocol (Falk et al.,1991; Seeger et al., 1999) using the HLA-A*02-specific antibody BB7.2,the HLA-A, —B, C-specific antibody W6/32, CNBr-activated sepharose, acidtreatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d. x 250 mm) packed with1.7 μm C18 reversed-phase material (Waters) applying a flow rate of 400nL per minute. Subsequently, the peptides were separated using atwo-step 180 minute-binary gradient from 10% to 33% B at a flow rate of300 nL per minute. The gradient was composed of Solvent A (0.1% formicacid in water) and solvent B (0.1% formic acid in acetonitrile). A goldcoated glass capillary (PicoTip, New Objective) was used forintroduction into the nanoESI source. The LTQ-Orbitrap massspectrometers were operated in the data-dependent mode using a TOP5strategy. In brief, a scan cycle was initiated with a full scan of highmass accuracy in the Orbitrap (R=30 000), which was followed by MS/MSscans also in the Orbitrap (R=7500) on the 5 most abundant precursorions with dynamic exclusion of previously selected ions. Tandem massspectra were interpreted by SEQUEST and additional manual control. Theidentified peptide sequence was assured by comparison of the generatednatural peptide fragmentation pattern with the fragmentation pattern ofa synthetic sequence-identical reference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose pancreatic cancer samples to a baseline of normaltissue samples. Presentation profiles of exemplary over-presentedpeptides are shown in FIGS. 1A-1AF. Presentation scores for exemplarypeptides are shown in Table 8.

TABLE 8 Presentation scores.  The table lists peptides that are very highly over-presented on tumors comparedto a panel of normal tissues (+++),highly over-presented on tumors compared to a panel of normal tissues (++) or over-presented on tumors compared to a  panel of normal tissues (+). SEQID Peptide No. Sequence Presentation   1 FVDTRTLL +++   3 ILIGETIKI +++  4 ALDPAAQAFLL +++   5 ALLTGIISKA +++   6 ALTGIPLPLI +++   7 ALVDIVRSL+++   8 ALYTGSALDFV +++  10 VLLDKIKNL +  11 ALYYNPHLL +++  12 AQYKFVYQV+++  14 FIIDNPQDLKV +++  15 FILANEHNV +++  16 GLIDYDTGI +++  17GLIDYDTGIRL ++  18 ALFVRLLAL +++  19 ALWHDAENQTVV +++  21 GLVDGRDLVIV+++  22 ILSTEIFGV +++  23 KLDSSGGAVQL ++  24 KLSENAGIQSL +++  25LINPNIATV +++  27 TLLAHPVTL +  29 YILPFSEVL +++  30 YIYKDTIQV +++  31YLDSMYIML ++  34 FLEDDDIAAV +++  35 FLFPSQYVDV +++  37 FLNPDEVHAI +  39FLTPSIFII +++  40 GLAPQIHDL +++  41 GLLAGNEKLTM ++  42 ILSDMRSQYEV +++ 43 HLGVKVFSV +++  44 ILAQVGFSV +++  45 ILYSDDGQKWTV +++  46 TMVEHNYYV+++  47 LIYKDLVSV +  48 LLDENGVLKL +++  49 LLDGFPRTV +++  50 LLFGSDGYYV+++  51 LLGPAGARA +++  52 LLSDPIPEV ++  53 LLWDPSTGKQV +++  54LTQPGPIASA +++  55 NLAPAPLNA +++  56 NLIGVTAEL +++  57 RLSELGITQA ++  58RQYPWGVVQV +++  59 SLSESFFMV +  60 SLWEDYPHV ++  61 SMYDGLLQA ++  62SVFPGARLL +++  63 SVTGIIVGV +++  64 TLFSEPKFAQV ++  67 VIWGTDVNV ++  68VLFDVTGQV +++  69 VLFSGSLRL +++  70 VLGVIWGV +++  71 VLLPEGGITAI +++  72VMASPGGLSAV +++  73 VMVDGKPVNL +  74 YIDKDLEYV +++  75 FSFVDLRLL +++  77RLFPGSSFL +++  79 VVYEGQLISI +  80 LLPGTEYVVSV +  81 VVYDDSTGLIRL +++ 82 ALIAEGIAL ++  83 ALSKEIYVI +++  85 FLSDGTIISV ++  86 GLGDFIFYSV + 88 IIDDTIFNL ++  90 KLLTPITTL +  91 LLFNDVQTL +  92 YLTNEGIAHL +  93SIDSEPALV +++  94 VMMEEFVQL +  95 ALADDDFLTV ++  96 ALAPATGGGSLLL +  98ALDQKVRSV +  99 ALESFLKQV + 100 ALFGAGPASI +++ 102 ALYPGTDYTV + 104FLQPDLDSL +++ 105 FLSEVFHQA + 106 FVWSGTAEA +++ 107 FVYGGPQVQL + 108IADGGFTEL +++ 109 ILASVILNV ++ 111 LLLAAARLAAA + 114 SLFPHNPQFI +++ 115SLMDPNKFLLL ++ 116 SMMDPNHFL ++ 118 TLWYRPPEL ++ 119 VLGDDPQLMKV + 120VLVNDFFLV ++ 122 MQAPRAALVFA + 123 KISTITPQI +++ 124 ALFEESGLIRI +++ 125ALLGKLDAINV +++ 126 ALLSLDPAAV +++ 128 ALYDVRTILL +++ 130 FLFGEEPSKL +131 FLIEEQKIVV +++ 132 FLWAGGRASYGV +++ 133 ILDDVSLTHL +++ 134ILLAEGRLVNL +++ 135 KLDDTYIKA +++ 136 KLFPGFEIETV +++ 137 KLGPEGELL +++138 NIFPNPEATFV ++ 140 SLLNPPETLNL +++ 142 SLYGYLRGA +++ 143 TADPLDYRL++ 144 TAVALLRLL +++ 145 TTFPRPVTV +++ 146 VLISGVVHEI +++ 147 YAFPKAVSV+++ 148 YLHNQGIGV + 149 ILGTEDLIVEV + 150 ALFQPHLINV ++ 151 ALLDIIRSL+++ 153 ALPKEDPTAV + 154 KVADLVLML + 155 LLLDPDTAVLKL ++ 156LLLPPPPCPA + 157 MLLEIPYMAA ++ 158 SLIEKYFSV + 159 SLLDLHTKV + 160VLLPDERTISL +++ 162 NADPQAVTM +++ 163 VMAPRTLVL ++ 164 YLGRLAHEV ++ 165YLLSYIQSI ++ 166 SLFPGQVVI +++ 167 MLFGHPLLVSV +++ 169 FMLPDPQNI +++ 171LLLDVTPLSL ++ 172 TMMSRPPVL ++ 173 SLAGDVALQQL +++ 174 TLDPRSFLL ++ 175ALLESSLRQA ++ 176 YLMPGFIHL +++

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalization, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0. Exemplary expression profiles of source genes of the presentinvention that are highly over-expressed or exclusively expressed inpancreatic cancer are shown in FIGS. 2A-2C. Expression scores forfurther exemplary genes are shown in Table 9.

TABLE 9 Expression scores. The table lists peptides from genes that arevery highly over-expressed in tumors comparedto a panel of normal tissues (+++), highlyover-expressed in tumors compared to a panelof normal tissues (++) or over-expressed intumors compared to a panel of normal tissues (+). SEQ ID Gene Gene Noname Sequence Expression   1 COL1A2 FVDTRTLL ++   2 COL1A2 FGYDGDFYRA ++  3 PTGS1, ILIGETIKI +++ PTGS2   6 CDK2 ALTGIPLPLI +   7 FADS3 ALVDIVRSL++   8 COL6A3 ALYTGSALDFV +   9 COL6A3 QIIDAINKV +  10 COL6A3VLLDKIKNL +  11 IPO7 ALYYNPHLL +  12 PTPN14 AQYKFVYQV +  18 TGFBIALFVRLLAL +++  24 RAI14 KLSENAGIQSL +  26 MAN2A1 SLYTALTEA +  31 ADAM9YLDSMYIML +  34 GFPT2 FLEDDDIAAV ++  38 TFPI2 FLTEAALGDA +  43 COL6A1HLGVKVFSV +++  44 SLC6A15 ILAQVGFSV ++  45 DCBLD2 ILYSDDGQKWTV ++  46DCBLD2 TMVEHNYYV ++  53 NLE1 LLWDPSTGKQV +  54 CXCL5 LTQPGPIASA +  56ARMC9 NLIGVTAEL ++  57 SHCBP1 RLSELGITQA +++  58 SEPT10, RQYPWGVVQV ++SEPT8, SEPT11  60 TRAM2 SLWEDYPHV ++  61 TRPV2 SMYDGLLQA ++  67 MCM4VIWGTDVNV +++  75 COL1A1 FSFVDLRLL +++  77 CREB3L1 RLFPGSSFL ++  79 FN1VVYEGQLISI +++  80 FN1 LLPGTEYVVSV +++  84 SLC1A4, FILPIGATV + SLC1A5 90 COL6A3 KLLTPITTL +  91 PLEC LLFNDVQTL +++  92 PLEC YLTNEGIAHL +++ 95 MCM4 ALADDDFLTV +++  99 PRKDC ALESFLKQV + 105 SERPINB2 FLSEVFHQA +113 GFPT2 LMMSEDRISL ++ 119 TAF6L VLGDDPQLMKV + 123 CDC27 KISTITPQI +124 CELSR3, ALFEESGLIRI + SLC26A6 126 PRKDC ALLSLDPAAV + 128 UBE2CALYDVRTILL + 132 HNRNPU FLWAGGRASYGV + 136 ASNS KLFPGFEIETV +++ 137SLC1A5 KLGPEGELL + 139 STAT2 SIDRNPPQL + 140 CCNA2 SLLNPPETLNL ++ 145NONO TTFPRPVTV + 146 MBTPS2 VLISGVVHEI + 151 FADS2 ALLDIIRSL ++ 153COPG1 ALPKEDPTAV + 165 NCAPG YLLSYIQSI +++ 166 POLA2 SLFPGQVVI + 173NCAPD2 SLAGDVALQQL + 175 CCND1 ALLESSLRQA +

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for 22 HLA-A*0201 restricted TUMAPsof the invention so far, demonstrating that these peptides are T-cellepitopes against which CD8+ precursor T cells exist in humans (Table10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nurnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 179) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.180), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+among CD8+T-cells and the percentage of specific multimer+cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Pancreatic Cancer Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIGS. 3A-3D together withcorresponding negative controls. Results for 4 peptides from theinvention are summarized in Table 10.

TABLE 10 In vitro immunogenicity of HLA class Ipeptides of the invention Exemplary results of in vitro immuno-genicity experiments conducted by the  applicant for HLA-A*02 restrictedpeptides of the invention. Results ofin vitro immunogenicity experimentsare indicated. Percentage of positive wells and donors (among evaluable) are   summarized as indicated <20% =+;  20%- 49% = ++; 50%-69% = +++; >=70% = ++++ SEQ Wells ID positive NoSequence [%]  17 GLIDYDTGIRL “+”  81 VVYDDSTGLIRL “+” 122 MQAPRAALVFA“++” 165 YLLSYIQSI “++” 167 MLFGHPLLVSV “++” 172 TMMSRPPVL “+” 173SLAGDVALQQL “+” 174 TLDPRSFLL “++” 119 VLGDDPQLMKV “+” 125 ALLGKLDAINV“+” 135 KLDDTYIKA “+++” 137 KLGPEGELL “+” 147 YAFPKAVSV “+” 148YLHNQGIGV “+++” 149 ILGTEDLIVEV “++” 156 LLLPPPPCPA “++”

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-μ2m for 1h at 37° C., washed again and detected with TMB solutionthat is stopped with NH2SO4. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

MHC class I binding scores. Binding of HLA-class I restricted peptidesto HLA-A*02:01 was ranged by peptide exchange yield: ≥10%=+; ≥20%=++;≥50=+++; >75%=++++

SEQ ID Peptide No Sequence exchange   1 FVDTRTLL “++”   2 FGYDGDFYRA“+++”   3 ILIGETIKI “+++”   4 ALDPAAQAFLL “+++”   5 ALLTGIISKA “+++”   6ALTGIPLPLI “+++”   7 ALVDIVRSL “+++”   8 ALYTGSALDFV “+++”   9 QIIDAINKV“++”  10 VLLDKIKNL “++”  11 ALYYNPHLL “++”  12 AQYKFVYQV “+++”  13FIDSSNPGL “++”  14 FIIDNPQDLKV “+++”  15 FILANEHNV “+++”  16 GLIDYDTGI“+++”  17 GLIDYDTGIRL “+++”  18 ALFVRLLAL “++”  19 ALWHDAENQTVV “+++” 20 GLIDIENPNRV “+++”  21 GLVDGRDLVIV “+++”  22 ILSTEIFGV “+++”  23KLDSSGGAVQL “++”  24 KLSENAGIQSL “++”  25 LINPNIATV “++”  26 SLYTALTEA“+++”  27 TLLAHPVTL “+++”  28 VLDEFYSSL “+++”  29 YILPFSEVL “++++”  30YIYKDTIQV “++”  31 YLDSMYIML “+++”  32 YVDDGLISL “++”  34 FLEDDDIAAV“++”  35 FLFPSQYVDV “+++”  36 FLGDLSHLL “++”  37 FLNPDEVHAI “++”  38FLTEAALGDA “+++”  39 FLTPSIFII “++”  40 GLAPQIHDL “++”  41 GLLAGNEKLTM“++”  42 ILSDMRSQYEV “+++”  43 HLGVKVFSV “++”  44 ILAQVGFSV “+++”  45ILYSDDGQKWTV “+++”  46 TMVEHNYYV “+++”  47 LIYKDLVSV “+++”  48LLDENGVLKL “+++”  49 LLDGFPRTV “++”  50 LLFGSDGYYV “+++”  51 LLGPAGARA“++”  52 LLSDPIPEV “+++”  53 LLWDPSTGKQV “+++”  54 LTQPGPIASA “+++”  55NLAPAPLNA “++”  56 NLIGVTAEL “++”  57 RLSELGITQA “+++”  58 RQYPWGVVQV“++”  59 SLSESFFMV “+++”  60 SLWEDYPHV “++++”  61 SMYDGLLQA “++”  62SVFPGARLL “+”  63 SVTGIIVGV “++++”  64 TLFSEPKFAQV “++++”  65 TLNEKLTAL“+++”  67 VIWGTDVNV “++++”  68 VLFDVTGQV “+++”  69 VLFSGSLRL “+++”  70VLGVIWGV “++++”  71 VLLPEGGITAI “+++”  72 VMASPGGLSAV “+++”  73VMVDGKPVNL “++++”  74 YIDKDLEYV “+++”  77 RLFPGSSFL “++++”  78SLQDTEEKSRS “+++”  79 VVYEGQLISI “+++”  80 LLPGTEYVVSV “+++”  81VVYDDSTGLIRL “+++”  82 ALIAEGIAL “+++”  83 ALSKEIYVI “+++”  84 FILPIGATV“++++”  85 FLSDGTIISV “++++”  86 GLGDFIFYSV “++++”  87 GLLPALVAL “+++” 88 IIDDTIFNL “+++”  89 KLADIQIEQL “+++”  90 KLLTPITTL “+++”  91LLFNDVQTL “+++”  92 YLTNEGIAHL “++++”  93 SIDSEPALV “+++”  94 VMMEEFVQL“+++”  95 ALADDDFLTV “+++”  96 ALAPATGGGSLLL “+++”  97 ALDDMISTL “+++” 98 ALDQKVRSV “++”  99 ALESFLKQV “+++” 100 ALFGAGPASI “+++” 101ALVEENGIFEL “+++” 102 ALYPGTDYTV “+++” 103 AVAAVLTQV “+++” 104 FLQPDLDSL“+++” 105 FLSEVFHQA “+++” 106 FVWSGTAEA “+++” 107 FVYGGPQVQL “+++” 109ILASVILNV “++++” 110 ILLTGTPAL “+++” 111 LLLAAARLAAA “+++” 112 LLSDVRFVL“+++” 113 LMMSEDRISL “+++” 114 SLFPHNPQFI “+++” 115 SLMDPNKFLLL “+++”116 SMMDPNHFL “++++” 117 SVDGVIKEV “+++” 118 TLWYRPPEL “+++” 119VLGDDPQLMKV “+++” 121 YLDEDTIYHL “++” 122 MQAPRAALVFA “++++” 123KISTITPQI “++” 124 ALFEESGLIRI “+++” 125 ALLGKLDAINV “+++” 126ALLSLDPAAV “++++” 127 ALSDLALHFL “++++” 128 ALYDVRTILL “+++” 129ALYEKDNTYL “+++” 130 FLFGEEPSKL “+++” 131 FLIEEQKIVV “+++” 132FLWAGGRASYGV “+++” 133 ILDDVSLTHL “++” 134 ILLAEGRLVNL “+++” 135KLDDTYIKA “+++” 136 KLFPGFEIETV “++++” 137 KLGPEGELL “+++” 138NIFPNPEATFV “+++” 139 SIDRNPPQL “+++” 140 SLLNPPETLNL “+++” 141SLTEQVHSL “+++” 142 SLYGYLRGA “+++” 144 TAVALLRLL “++” 145 TTFPRPVTV“+++” 146 VLISGVVHEI “+++” 147 YAFPKAVSV “++” 148 YLHNQGIGV “++” 149ILGTEDLIVEV “+++” 150 ALFQPHLINV “++++” 151 ALLDIIRSL “++++” 152ALLEPEFILKA “++++” 153 ALPKEDPTAV “+++” 154 KVADLVLML “+++” 155LLLDPDTAVLKL “+++” 156 LLLPPPPCPA “+++” 157 MLLEIPYMAA “+++” 158SLIEKYFSV “++++” 159 SLLDLHTKV “+++” 160 VLLPDERTISL “++++” 161YLPDIIKDQKA “+++”

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The invention claimed is:
 1. An expression vector expressing a nucleicacid encoding a peptide consisting of the amino acid sequence SEQ ID NO:14.
 2. A recombinant host cell comprising the expression vector of claim1, wherein said host cell is a dendritic cell or a macrophage.
 3. Amethod for producing a peptide consisting of the amino acid sequence SEQID NO: 14, comprising culturing the host cell of claim 2, and isolatingthe peptide from the host cell or its culture medium.
 4. An in vitromethod for producing activated antigen-specific CD8+ cytotoxic Tlymphocytes, comprising contacting in vitro T cells with antigen loadedhuman HLA-A*02 expressed on the surface of a suitable antigen-presentingcell or an artificial construct mimicking an antigen-presenting cell fora period of time sufficient to activate said T cells, wherein saidantigen is a peptide consisting of the amino acid sequence SEQ ID NO:14.
 5. The method of claim 4, wherein the T cells are autologous to thepatient.
 6. The method of claim 4, wherein the T cells are obtained froma healthy donor.
 7. The method of claim 4, wherein the T cells areobtained from tumor infiltrating lymphocytes or peripheral bloodmononuclear cells.
 8. The method of claim 4, wherein the activated Tcells are expanded in vitro.
 9. The method of claim 4, wherein theantigen presenting cell is infected with recombinant virus expressingthe peptide.
 10. The method of claim 9, wherein the antigen presentingcell is a dendritic cell or a macrophage.
 11. The method of claim 8,wherein the expansion is in the presence of an anti-CD28 antibody andIL-12.
 12. A pharmaceutical composition comprising a peptide consistingof the amino acid sequence SEQ ID NO: 14 in the form of apharmaceutically acceptable salt and an immune-stimulating adjuvant. 13.The pharmaceutical composition of claim 12, wherein the pharmaceuticallyacceptable salt is chloride salt.
 14. The pharmaceutical composition ofclaim 12, wherein the pharmaceutically acceptable salt is acetate salt.15. The pharmaceutical composition of claim 12, wherein the adjuvant isselected from anti-CD40 antibody, imiquimod, resiquimod, GM-CSF,cyclophosphamide, interferon-alpha, interferon-beta, CpGoligonucleotides and derivatives, poly-(I:C) and derivatives, RNA,sildenafil, particulate formulations with poly(lactide co-glycolide)(PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13,IL-15, IL-21, and IL-23.
 16. The pharmaceutical composition of claim 12,wherein the adjuvant comprises IL-2.
 17. The pharmaceutical compositionof claim 12, wherein the adjuvant comprises IL-7.
 18. The pharmaceuticalcomposition of claim 12, wherein the adjuvant comprises IL-15.
 19. Thepharmaceutical composition of claim 12, wherein the adjuvant comprisesIL-21.
 20. The pharmaceutical composition of claim 12, wherein theadjuvant comprises cyclophosphamide.